Cell Biochem Biophys (2014) 69:681–691 DOI 10.1007/s12013-014-9853-3
ORIGINAL PAPER
Differential Sensitivity of Telomerase from Human Hematopoietic Stem Cells and Leukemic Cell Lines to Mild Hyperthermia Abdolkhaleg Deezagi
Published online: 4 March 2014 Ó Springer Science+Business Media New York 2014
Abstract We have investigated the effects of hyperthermia (HT) on cell proliferation and telomerase activity of human hematopoietic stem cells (HSCs) and compared with human leukemic cell lines (TF-1, K562 and HL-60). The cells were exposed to HT at 42 and 43 °C up to 120 min. The cells were incubated at 37 °C for 96 h. Then the cells were collected and assayed for cell proliferation, viability, telomerase activity, and terminal restriction fragment (TRF) lengths. The enzyme activity from HSCs was decreased up to 68.6 at 42 and 85.1 % at 43 °C for 120 min. This inhibition in leukemic cells was up to 28.9 and 53.6 % in TF-1; 53 and 63.9 % in K562; 45.2 and 61.1 % in HL-60 cells. The treated cells showed TRF lengths about 5.3 kb for control HL-60 cells, 5.0 kb for HL-60 cells treated at 42 and 4.5 kb at 43 °C for 120 min. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0–4.5 kb for treated cells at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. TRF length was constant at treated two types of cells, which implies that in cells subjected to mild HT no telomere shortening was observed. Keywords Hyperthermia Telomerase activity Cell proliferation Hematopoietic stem cells Human leukemic cell lines
A. Deezagi (&) Department of Biochemistry, National Institute of Genetic Engineering and Biotechnology, Km. 17, Karaj-Tehran Freeway, Pajouhesh Blvd., P.O.Box 14155-6343, Tehran, Iran e-mail: [email protected]
Abbreviations HT Hyperthermia TRAP Telomeric repeat amplification protocol assay HSCs Hematopoietic stem cells TRF Terminal restriction fragment
Introduction Heat stress causes a variety of alteration in cellular physiology. One of the well-documented effects of the exposure of mammalian cells to elevated temperatures are the inhibition of macromolecules synthesis, including DNA, RNA and protein synthesis, autophagy, and protein aggregation [1, 2]. Macromolecules, in particular polypeptides, have differential sensitivity to heat stress due to their specific heat capacity [3]. In several humans, hematopoietic and leukemic cell lines exposure to hyperthermia (HT) lead to morphological maturation, repressed cell proliferation and reduced clonally cell growth. These phenotypic changes are linked to change in expression of important growth regulating genes, growth factors or their receptors which finally block the cell division [4]. The human embryo and fetus may be especially vulnerable to chemical and physical insults during defined stages of development. In particular, the scheduled processes of cell proliferation, cell migration, cell differentiation, and apoptosis that occur at different times for different organ structures can be susceptible to elevated temperatures [5]. Existing data on the differential heat sensitivity of normal and malignant hematopoietic cells have encouraged attempts to apply HT as a potential purging agent in the removal of leukemic cells from the donor bone marrow inoculums prior to
123
682
autologous transplantation and novel therapeutic anti-cancer strategies [6–8]. The exact molecular mechanism of the differential sensitivity of leukemic and normal hematopoietic stem cells (HSC) to HT has not been understood. In the previous work, we have showed that the telomerase activity was significantly reduced in three different human leukemic cells (K562, HL-60 and TF-1 cells) in response to HT [9]. Telomeres are the molecular caps at the ends of chromosomes that are composed of repetitive TTAGGG sequences and associated proteins [10]. Telomeres become shorter during somatic development and with the increasing age of the individual [11]. The maintenance of telomeres throughout many cycles of cell division requires the enzyme telomerase, which consists of two core components, the RNA-subunit, hTR, containing the template, and a catalytic protein subunit, human Telomerase Reverse Transcriptase (hTERT) [12]. It has been proposed that telomerase is repressed in somatic tissues, and that telomeres become shorter during somatic development and with the increasing age of the individual [13]. In contrast, leukemic and cancerous cells are characterized by an unlimited proliferative potential, and accumulating evidence suggests that the reactivation of telomerase is a critical step in carcinogenesis and tumorogenesis; also telomerase activity was detected in nearly 90 % of various human tumor samples [14, 15]. Unlike normal somatic cells, telomerase activity has been detected at low levels in hematopoietic progenitor cells, which is up regulated in response to cytokine stimulation [16, 17]. The level of telomerase is low in the majority of human stem cells, whereas it is up regulated in cells that undergo rapid expansion, such as committed hematopoietic progenitor cells, activated lymphocytes, or keratinocytes, even within tissues with a low cell turnover such as the brain [18]. Most cycling HSCs display telomerase activity [19]. Following an initial increase, telomerase activity is down regulated as HSCs, and progenitor cells proliferate and differentiate into more mature cells that display low to negligible levels of telomerase activity [20, 21]. The exact molecular mechanism of the differential sensitivity of leukemic and normal HSCs to HT has not been understood. In this research, we have investigated the telomerase activity and cell proliferation of human HSCs and human leukemic cell lines in response to mild HT (42–43 °C). For this purpose, HSCs were isolated from human cord blood (CB) by immuno-magnetic separation technique. HSCs and human leukemic cell lines were exposed to HT at 42 °C up to 120 min. Then, the cells were incubated at 37 °C for 96 h. Finally, the cells were collected and assayed for cell proliferation, viability, telomerase activity, and telomere length.
123
Cell Biochem Biophys (2014) 69:681–691
Material and Method Human leukemic Cell Culture TF-1 human cell line was purchased from DSMZ Company (DSMZ, Braunschweig, Germany). HL-60 and K562 cells were purchased from American Type Culture Collection (ATCC) (ATCC, Rockville, MD, USA). The Cells were maintained at 37 °C, 5 % CO2 in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10 % FBS (Gibco, Paisley, UK), 120 mg/l penicillin, and 200 mg/l streptomycin. In addition, the culture medium of TF-1 cells was supplemented with 2 ng/ml of rhGM-CSF (Roche, Mannheim, Germany). Cells were cultured at cell density about 2–5 9 105 cells/ml for 4 days. Cell viability was determined by trypan blue-dye exclusion test. Cord Blood Collection Umbilical CB to be discarded were collected. CB was obtained from informed and consenting donors at the Mustafa Khomeini Hospital, Tehran-Iran. The umbilical cord was clamped according to standard hospital procedure, and CB collections were performed ex utero. CB was collected into a sterile collection 50 ml falcon tubes containing 10 ml of Citrate Phosphate Dextrose solution (Sigma, St. Louis, MO, USA). The collection volume varied between 15 and 40 ml. After fourfold dilution with Ca??/Mg?? free Hanks, Balanced Salt Solution (HBSS) (Sigma, St. Louis, MO, USA), mono-nuclear cells (MNCs) were isolated by density gradient using 10 ml of FicollPaque reagent (Nycomed, Oslo, Norway) and 30 ml of diluted CB. The two-phase system was centrifuged at 4009g for 40 min without break, at 20 °C in a swingingbucket rotor (Beckman Coulter Inc., CA, USA). MNCs, collected from the interface of the two phases, were washed twice with PBS. MNCs were counted by hemocytometer neobar, and cells’ viability was assayed by trypan blue dye exclusion test. Separation of CD34?/CD133? Cells Enrichment of HSCs in CB is based on the expression of certain surface antigens, such as CD34 and CD133, or on the lack of expression of lineage-specific antigens. Immunomagnetic negative selection kit (StemSep StemCell Technologies, Vancouver, Canada) was used for the isolation of CD34? or CD133? cells using paramagnetic microbeads conjugated to specific monoclonal antibodies. In negative selection of lineage-negative (Lin-) cells, the unwanted cells are labeled with antibodies against known markers for mature hematopoietic cells (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A)
Cell Biochem Biophys (2014) 69:681–691
and retained in the column. Unlabeled cells pass through the column and are collected as the Lin- cell fraction. To enrich progenitor cells, lineage-committed cells were depleted [22, 23]. The conditions resulting in optimal separation of Lin- cells were 100 ll of Progenitor Enrichment Cocktail and 60 ll of magnetic iron particles per 8 9 107/ml MNCs, as recommended by the manufacturer. MNCs (8 9 107/ml) were labeled with Progenitor Enrichment Cocktail (100 ll/ml) containing antibodies against CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and Glycophorin A (StemCell Technologies, Vancouver, Canada) at room temperature for 15 min. Subsequently, the cell suspension was incubated with magnetic iron particles (60 ll/ml) at room temperature for 15 min. Cell suspension was loaded into MACS LD column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and unlabeled cells passing through the column were collected (Lin- fraction). The column was then washed twice with 1 ml of buffer, and the remaining Lin? cells were collected for control purposes. The isolation method and characterization was carefully described in our last paper [24]. The purity of isolated CD34? HSCs was assayed according the recommendation of the HSC Immunomagnetic negative selection kit (StemSep StemCell Technologies, Vancouver, Canada). Purity was measured using flow cytometery by staining with a FITCconjugated anti-human CD34 antibody catalog number 10413 (StemSep StemCell Technologies, Vancouver, Canada). The cell’s numbers and cell’s viability were determined by a hemocytometer, using the Trypan blue test. The purity of CD34 for suspension culture and various assays, isolated cells were cultured in RPMI-1640 medium (Gibco, Paisley, UK) that supplemented by FCS 20 % with combination of the following cytokines; recombinant human erythropoietin (rhEPO) 6 U/ml (Roche, Mannheim, Germany), recombinant human interleukine-3 (rhIL-3) 50 ng/ml (Sandoze, Basel. Switzerland), recombinant human Granulocyte–Macrophage colony stimulating factor (rhGM-CSF) 20 ng/ml (Sandoze, Basel, Switzerland) and rhIL-6, 20 ng/ml (Sandoze, Basel, Switzerland). Heat Treatment Heat shock was applied at 41–44 °C for different intervals of 15, 30, 60, and 120 min for leukemic cells. Because of limitation in the providing of HCSs, the HSCs were treated only at 42 and 43 °C for 15, 30, 60, and 120 min. The leukemic cells and HSCs (106 cells/ml in RPMI 1640) were separately treated by HT by immersion in thermostated water bath (Memert, Braunschweig, Germany) with ±0.1 °C precision. Control cells were similarly treated at 37 °C. At the end of heat treatment, the cells were allowed to stand for 5 min at room temperature. Cell viability was
683
immediately determined, and 2 9 105 cells were stored for telomerase assay at -20 °C in order to assay the short time effect of HT on the telomerase activity before incubation. Then, the remained portion of the treated cells were transferred to the 6-wells plates (NUNC, Roskilde, Denmark) and cultured in RPMI and 10 % FCS at final cell density of 2 9 105 cells/ml. Cells were incubated at 37 °C for 96 h. Then, the cells were collected and assessed for cell proliferation, telomerase activity, and telomere length as described below. Cell Growth and Proliferation Assays In order to examine the effect of heat shock on the cell growth and cell proliferation, cells were collected after 96 h and the total cell number, viability, and MTT cell proliferation assay were done as below. Cell count and viability were enumerated using a Neobar hemocytometer. MTT Assay After 96 h of incubation, 100 ll of finely resuspended control and heat shock-treated cells were transferred to flat bottom 96 microtiter plates. Then 10 ll of freshly prepared (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, U.S.A.) solution (5 mg/ml in PBS) was added to each well and were incubated for 4 h. Finally, 50 ll of MTT lysis solution (20 % sodium dodecyl sulfate w/v and 50 % dimethyl formamide v/v) was added to each well and incubated overnight. Absorbance was read at 620 nm using an ELISA reader. TRAP-PCR ELISA Assay The telomerase activity was determined using a telomerase repeat amplification protocol TRAP-PCR-ELIZA kit (Roche, Mannheim, Germany) which described by Keith and Monaghan [25] for the first time. This method allows highly specific amplification of telomerase-mediated elongation products combined with nonradioactive detection following an ELISA protocol. The assay separated in two steps. In the first step, telomerase adds telomeric repeats (TTAGGG) to the biotin-labeled synthetic P1TSprimer, and then, the elongation product are amplified using the primers P1-TS. In the second step, PCR product is denatured and hybridized to a digoxigenin-(DIG)— labeled, telomeric repeat-specific detection probe. The resulting product is immobilized via the biotin-labeled primer to a coated microplate. The immobilized PCR product is then detected with an antibody against DIG-POD that is conjugated to peroxidase. Finally, the probe is visualized by virtue of peroxidase metabolizing TMB. Briefly, 2 9 104 cells were washed with cold PBS and
123
684
Cell Biochem Biophys (2014) 69:681–691
lysed in 200 ll of precooled lysis solution and incubated on ice for 30 min, then centrifuged at 16,0009g for 20 min at 4 °C. For PCR amplification, 2 ll of supernatant and 25 ll of reaction mixture (containing Telomerase substrate, primers, nucleotides, and Taq polymerase) were transferred into a suitable tube, and then sterile water was added to a final volume of 50 ll. An amplification reaction was carried out by PCR. The 5 ll amplification product was mixed with 20 ll of denaturation reagent and incubated at room temperature for 10 min, and 225 ll of hybridization buffer was added and mixed thoroughly, then 100 ll of mixture per well was transferred into the precoated microtiter plate and incubated at 37 °C on a shaker for 2 h. Anti-DIC-POD working solution (100 ll) was added and incubated at room temperature for 30 min with shaking. The solution was removed completely, and the precipitate was rinsed five times with 250 ll of washing buffer per well for a minimum of 30 s. After removing the washing buffer, 100 ll of TMB substrate solution was added and incubated for color development at room temperature for 20 min with gentle shaking. Finally, 100 ll of stop reagent was added to each well to stop color development. The amount of TRAP products was determined by the measurement of the absorbance at 450 and 690 nm. A negative as well as a positive control was run each time. A negative control was provided for each extract by heat inactivating the telomerase enzyme present in cell lysate at 65 °C for 10 min prior to the PCR step. Southern Blot Analysis of Telomere Length and Terminal Restriction Fragment (TRF) Length Genomic DNA of the heat-treated HL-60 and HSCs cells was isolated using High Pure Template Preparation kit (Roche, Mannheim, Germany), and TRF length was estimated using Telo TAGGG Telomere Length Assay Kit (Roche, Mannheim, Germany). In brief, 2 lg of genomic DNA was digested with restriction enzymes HinfI and RasI and separated on 0.8 % w/v gel. The DNA fragments were then transferred to a positively charged nylon membrane (Whatman Oxon, UK) in 209 SSC buffer overnight at room temperature. The membrane were hybridized with a digoxigenin (DIG)-labeled telomere-specific probe, and detected by an anti-DIG-alkaline phosphatase and CDPStar as the chemiluminescent substrate. Statistical Analysis Each experiment was performed three times for all data, each carried out in duplicated sequences. Data were analyzed using a one-way analysis of variance (ANOVA) values were given as the mean ± standard deviation (SD), and biological variables were compared using the Students’
123
Fig. 1 Purity of CD34? cells was measured using flow cytometery by staining with a FITC-conjugated anti-human CD34 antibody. The single-parameter histogram and frequency of CD34? cells, a in the started CB, b in the finally enriched and isolated fractions
t test. By convention, a a-level of p \ 0.05 was considered to be statistically significant. Finally, the correlation between heat shock and the telomerase activity, cell proliferation was calculated statistically.
Results HSCs Isolation and Purity Assay CBs were provided from more than 10 newborns. The total cell’s numbers from each step of isolation were counted. Purity of CD34? cells was measured using flow cytometery by staining with a FITC-conjugated anti-human CD34 antibody. The percent of finally isolated total CD34? to started total mononuclear cells was about 1.87 ± 0.61 %. The frequency of CD34? cells in the started CBs were
Cell Biochem Biophys (2014) 69:681–691 1.6
*
95
%of viability
85
*
75
*
65
*
55 45 35
Absorbance at 620 nm
105
685
1.4
*#
1.2
=
1
& 0.8
&
0.6
&
0.4
*= &
0
0
15
30
60
90
120
0
Heat shock time (minutes)
15
30
85
=86#
55
=
#
= =
#
45
#
35
#
25 0
15
30
60
90
120
Heat shock time (minutes) 43 C before incubation
42 C before incubation
43 C after incubation
42 C after incubation
(b) Fig. 2 The effect of heat shock on the cell viability of human eryhtroleukemic TF-1 and human HSCs. The cells were treated by HT (42 and 43 °C up to 120 min), and the cells’ viability was immediately assayed after HT or before incubation. The cells were incubated for 96 h. Then the cells were collected, and the cell viability was detected after incubation. a The TF-1 cells’ viability b human HSCs’ viability. The results are mean ± 1 SEM for four separate experiments, *p \ 0.01 compared untreated versus treated TF-1 cells at 43 °C for 0, 15, 30, 60, 90, and 120 min after 96 h incubation, #p \ 0.01 compared untreated and treated human HSCs cells at 43 °C for 0, 15, 30, 60, 90, and 120 min after 96 h incubation, =p \ 0.05 compared to untreated and treated human HSCs cells at 42 °C for 0, 60, 90, and 120 min after 96 h incubation. In other condition, significant differences were not observed
about 0.9–1.9 % (Fig. 1a) and 51–69 % in the final enriched fractions (Fig. 1b). Cellular Viability and Cell Proliferation After Heat Shock To assess the inhibitory effects of HT on the cell growth, cytotoxic effects of HT on cells was determined using trypan blue exclusion test, cell counting, and MTT cell proliferation assay. The effect of HT on the cell viability was immediately assayed after heat treatment or before incubation and after 96 h incubation. HT caused both
Cell proliferation (% of Controls
95
#
60
120
(a)
(a)
65
45
Heat Shock time (minutes)
105
%of viability
&
0.2
25
75
#
&
120 100 80 60
42 C TF-1 43 C TF-1
40
42 C HSC
20
43 C HSC
0 0
15
30
45
60
120
Heat Shock time (minutes)
(b) Fig. 3 The effect of HT on the cell proliferation of human eryhtroleukemic TF-1 and human HSCs. The cells were treated by HT (42 and 43 °C up to 120 min) and were incubated for 96 h at 37 °C. Then, the cells proliferation was assayed by MTT assay. a Absorbance at 620 nm from MTT assay of the cells. b Percent of cell proliferation of treated cells in comparison to untreated control cells. Values are the means ± 1 SEM of data from duplicate cultures for four separate experiments (n = 4). *p \ 0.01 compared untreated and treated TF-1 cells at 43 °C for 0 and 120 min. =p \ 0.05 compared treated TF-1 cells at 43 °C for 15 and 120 min after 96 h incubation, #p \ 0.01 compared to untreated and treated TF-1 cells at 43 °C for 0 and 60. &p \ 0.01 compared untreated and treated human HSCs cells at 43 °C for 0, 15, 30, 60, 90, and 120 min. In other condition, significant differences were not observed
short- and long-term damages in leukemic cell lines and HSC cells. Short-term damage was expressed as a decrease in cell viability immediately after HT and before incubation. TF-1 cells were viable ([89 %) after 120 min heating at 42 and 43 °C (Fig. 2a). HSCs were sensitive to HT in comparison to TF-1 cells; the viability was decreased in HSCs up to 74.5 ± 3.3 and 62.3 ± 5.9 % at 42 and 43 °C for 120 min HT (Fig. 1b). Long-term damage was expressed when the cells were incubated at 37 °C for 96 h after HT. 120 min of heating at 42 and 43 °C dropped the TF-1 cell viability to 79.8 ± 1.7 and 46.7 ± 10.2 % (Fig. 1a); these condition caused a higher decrease in HSCs viability 64.8 ± 3.8 and 37.6 ± 7.1 %, respectively
123
686
Cell Biochem Biophys (2014) 69:681–691
Fig. 4 Telomerase activity of human leukemic cells and HSCs after treatment by HT. The cells were treated by HT (42 and 43 °C up to 120 min) and telomerase activity was immediately determined after HT using the TRAP-PCR–ELISA method as described in methods. a Telomerase activity after HT at 42 °C (absorbance at 450 nm). b The percent of telomerase activity in comparison to untreated control cells after HT at 42 °C. c Telomerase activity after HT at 43 °C (Absorbance at 450 nm). d The percent of telomerase activity in comparison to untreated control cells after HT at 43 °C. (n = 4), and values are the means ± 1 SE of data from at least two independent experiments. *p \ 0.01 compared untreated and treated
HSCs cells at 42 °C for 0, 15, 30, 60, and 120 min after 96 h incubation. ?p \ 0.01 compared treated leukemic cells at 42 °C for 15, 60, and 120 min after 96 h incubation. #p \ 0.01 compared untreated and treated HSCs cells at 42 °C for 0, 15, 30, 60, and 60 after 96 h incubation. ?p \ 0.01 compared untreated and treated leukemic cells at 43 °C for 0, 15, 30, 60, 90, and 120 min. **p \ 0.01 compared treated leukemic cells at 43 °C for 15 and 120 min. &p \ 0.01 compared treated versus untreated HSCs cells at 43 °C for 0, 15, 30, 60, and 120 min. &&p \ 0.01 compared treated HSCs cells at 43 °C for 15 and 120 min. In other condition, significant differences were not observed
(Fig. 2b). The cell proliferation and growth rate of heat shock-treated cells were assessed using colorimetric MTT cell proliferation assays at each temperature and time points. The cells were treated by HT and cultured (10,000 cell/well in 100 ll in flat bottom 96 wells) at 37 °C for 96 h. Then MTT dye was added, and absorbance was measured. The values for two types of cells were averaged, and growth curves were constructed. The values for untreated control cells were 1.18 ± 0.24 absorbance unit (AU) for TF-1 cells and 0.72 ± 0.12 AU for HSCs (Fig. 3a). The results of the MTT cell proliferation assay indicated to the low potency of HSCs in comparison to TF1 cells. Results of the HT-treated cells indicated that the cells proliferation was significantly decreased with the increasing of time and temperature in both type of cells. When the cells were treated by HT up to 120 min, the cell
proliferation potency was decrease up to 40 % (0.433 ± 0.048 AU) for HSCs and up to 30 % (0.824 ± 0.171 AU) for TF-1 cells in comparison to untreated control cells at 42 °C for 120 min. Treatment of cells by HT at 43 °C caused a sharp reduction of cell proliferation up to 71 % (0.211 ± 0.02 AU) for HSCs and 52 % (0.56 ± 0.12 AU) for TF-1 cells (Fig. 3b).
123
Effect of HT on Telomerase Activity To examine the effect of heat shock on telomerase activity, the activity of enzyme was assayed by TRAP-PCR ELISA method as described in methods. The telomerase activity was analyzed immediately and over 96 h incubation after heat shock. When the cells were treated by HT and telomerase activity was assayed before incubation, the enzyme
Cell Biochem Biophys (2014) 69:681–691
687
Fig. 5 Telomerase activity of human leukemic cells and HSCs after treatment by HT and incubation for 96 h. The cells were treated by HT (42 and 43 °C up to 120 min) and were incubated for 96 h at 37 °C. Telomerase activity was determined using the TRAP-PCR– ELISA method as described in methods. a Telomerase activity after HT at 42 °C (absorbance at 450 nm). b The percent of telomerase activity in comparison to untreated control cells after HT at 42 °C. c Telomerase activity after HT at 43 °C (absorbance at 450 nm). d The percent of telomerase activity in comparison to untreated control cells after HT at 43 °C. (n = 4), and values are the
means ± 1 SE of data from at least two independent experiments. *p \ 0.01 compared to untreated versus treated leukemic cells at 42 °C for 0, 30, 60, and 120 min. #p \ 0.01 compared to treated HSCs cells at 43 °C for 0, 15, 30, 60, and 120 min after 96 h incubation. ##p \ 0.01 compared to untreated and treated leukemic cells at 43 °C for 0, 15, 30, 60, and 120 min after 96 h incubation. **p \ 0.01 compared to treated and untreated HSCs cells at 43 °C for 0, 15, 30, 60, and 120 min after 96 h incubation. In other condition, significant differences were not observed
activity from HSCs was reduced up to 58 % at 42 °C for 120 min (0.260 ± 0.018 AU for control cells, 0.109 ± 0.016 at 42 °C for 120 min). This reduction in leukemic cells was about 47.3 % in TF-1 cells (0.701 ± 0.002 and 0.370 ± 0.057 AU), 30.5 % in K562 cells (0.685 ± 0.042 and 0.544 ± 0.029 AU), and 31.4 % in HL-60 cells (0.617 ± 0.064 for control cells and 0.426 ± 0.082 AU for treated cells at 42 °C for 120 min), respectively, Fig. 4a, b. The enzyme activity from HSCs was reduced up to 73.1 % at 43 °C for 120 min (0.260 ± 0.018 AU for control cells and 0.072 ± 0.009 AU at 43 °C for 120 min). This reduction in leukemic cells was about 52.2 % in TF-1 cells (0.701 ± 0.002 and 0.355 ± 0.011 AU), 35.9 % in K562 cells (0.679 ± 0.021 and 0.435 ± 0.028 AU), and 37.7 % in HL-60 cells (0.617 ± 0.06 4 for untreated control cells and 0.405 ± 0.058 AU for treated cells at 43 °C for 120 min), respectively, Fig. 4c, d.
When the cells were incubated 96 h at 37 °C after HT, the telomerase activity of HSCs was significantly reduced in comparison to leukemic cells. The enzyme activity from HSCs was decreased up to 68.6 at 42 °C for 120 min (0.24 ± 0.014 for control cells and 0.076 ± 0.024 AU at 42 °C for 120 min). This inhibition in leukemic cells was up to 28.9 in TF-1 cell (0.780 ± 0.058 and 0.573 ± 0.036 AU), 53 % in K562 cells (1.126 ± 0.107 and 0.529 ± 0.15 AU), and 45.2 % in HL-60 cells (0.991 ± 0.024 for untreated control cells and 0.543 ± 0.036 AU for treated cells at 42 °C for 120 min), respectively at that conditions (Fig. 5a, b). The enzyme activity from HSCs was decreased up to 85.1 % at 43 °C for 120 min (0.24 ± 0.014 for control cells and 0.035 ± 0.005 AU at 43 °C). This inhibition in leukemic cells was up to 53.6 % in TF-1 cells (0.780 ± 0.058 and 0.35 ± 0.033 AU), 63.9 % in K562 cells (1.124 ± 0.126 and 0.405 ± 0.074 AU), and 61.1 % in HL-60 cells (0.897 ± 0.039 and
123
688
Fig. 6 Southern blot analysis of the effects of HT on telomere length. The HL-60 and HSCs cells were exposed to HT at 42 and 43 °C for 120 min. Then, the cells were incubated at 37 °C for 96 h, and genomic DNA was extracted and analyzed as described in methods for telomere length. The TRF length of DNA from the treated and control cells were determined. MW molecular weight DNA Marker, P positive control, N negative control, lane 1 untreated HL-60 cells; HL-60 cells treated by HT at 42 °C (lane 2) and 43 °C (lane 3) for 120 min; lane 4 untreated HSCs cells; HSCs cells treated by HT at 42 °C (lane 5) and 43 °C (lane 6) for 120 min
0.351 ± 0.034 AU), respectively at that conditions (Fig. 5c, d). The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. Effect of Heat Treatment on Telomere Length and TRF Length Southern blot analysis was carried out after treatment of cells at 42 and 43 °C for 120 min and 96 h incubation at 37 °C. Figure 6 reveals that the pattern of TRF length did not show a significant difference in heat-treated HL-60 cells at 42 and 43 °C in comparison with the untreated control cells (lanes 1, 2, 3). The treated cells showed that the TRF lengths of untreated HL-60 cells ranged approximately from 3.2 to 7.4 kb, with a mean value of 5.3 kb, averaged 5.0 kb for 120 min at 42 °C and 4.5 kb for 120 min at 43 °C. These results were constant at treated HL-60 cells which implies that in cells subjected to HT at 42 and 43 °C for 120 min no telomere shortening was observed. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0–4.5 kb for treated cells at 42 and 43 °C for 120 min (lanes 4, 5 and 6). In this cell, we did not observed significant telomere shortening too.
Discussion Here, we analyzed short-term effects of mild HT to evaluate telomerase activity and the rate of sensitivity of
123
Cell Biochem Biophys (2014) 69:681–691
hematopoietic cells. According the previous reported data about the high sensitivity of leukemic cells to HT, our cell survival and cell proliferation data indicated to the hyperthermic sensitivity of leukemic cells in comparison to HSCs too. In spite of hyperthermic sensitivity of leukemic cells, we provide evidence that the telomerase activity of HSCs is more sensitive to HT than leukemic cell lines. The mechanism that induces immediate decrease of telomerase activity did not understand. So it seems that the primitive decrease in telomerase activity is related to immediate effect of HT in inactivating enzyme [9]. The presence of telomerase activity in hematopoietic stem/progenitor cells in comparison to the majority of human somatic cells implies an important function. While telomerase expression may serve to delay telomere shortening in these cells, telomere attrition is not prevented. It is beginning to emerge that telomerase may play an additional role to telomere maintenance in hematopoietic cell function [26]. The expanding development of specific telomerase inhibitors for the treatment of leukemia and cancer by induction of apoptosis, including hematopoietic malignancies, necessitates the urgent clarification of telomerase function in hematopoietic cells and malignancies [27, 28]. It is crucial that the role of telomerase in both normal hematopoiesis and leukemia is understood, allowing us to determine the potential effects of clinical inhibition or activation of telomerase on HSC function [29]. Agrawal et al. previously presented that moderate HT (43 °C) enhances telomerase activity. Inhibition of telomerase activity with human telomerase RNA-targeted antisense agents, and in particular GRN163L, results in enhanced HT-mediated IR-induced cell killing, and ectopic expression of catalytic unit of telomerase (TERT) [30]. Our findings were not in agreement with their results. For explanation and clear discussion, it must be considered that the type of the cells used by us is different from that of theirs. Agrawal et al. used 293, HeLa, GM857, and primary fibroblast cell lines (GM5823, HFF, and BJ) with their corresponding stably transfected hTERT counterparts (GM5823?hTERT, BJ?hTERT, and HFF?hTERT) Tercnull and Tert-null mouse embryonic cells along with their wild-type counterparts. They used multiple approaches to determine the effect of telomerase inactivation by heat. They concluded interestingly, heat does not induce any telomerase activity in primary fibroblasts (BJ or GM5823) and, when treated with telomerase inhibitors, did not show any major influence on clonogenic survival after heat shock and IR treatments compared with the telomerase-positive cells [30]. Therefore, their finding in primary cells and wild type cancerous cells indicated to the inhibition of telomerase activity by HT at 43 °C for 1 h. But in over expression systems with transfection of these cells by hTERT, they showed telomerase activity was increased. For clear
Cell Biochem Biophys (2014) 69:681–691
discussion of telomerase activation by HT in over expression systems, it must consider that, viral vectors and transfection systems used for cancer gene therapy are usually delivered by direct intratumoral administration, and HT can affect the transfection and expression efficiency in those systems. Siddiqui et al. [31] studied the role of HT in vitro and in vivo in an attempt to achieve higher transfection rates (especially, larger volume of spread). They concluded that, in the in vitro studies, A549 cells infected with the adenoviral construct did not show any difference in gene expression level in the presence or absence of HT. In vivo, the effect of HT on the volume of gene expression in A549 tumors was highly variable with some groups of mice showing better spread in the presence of HT and others showing reduced spread with HT [31]. Ito et al. recently reported that heat-inducible transgene expression with transcriptional amplification mediated by a transactivator. They have combined heat inducibility with highlevel transgene expression, developed a heat-inducible transgene expression system with transcriptional amplification mediated by a tetracycline-responsive transactivator [32]. Zintchenko et al. [4] have shown the temperature dependency of gene expression which induced by PNIPAM-based copolymers and the potential of HT in gene transfer too. Our data significantly show that the telomerase of HSCs are more sensitive to heat treatment than malignant hematopoietic and leukemic cells, but we did not observe a significant telomere shortening in these cells after treatment by HT at 42 and 43 °C for 120 min. In one study, telomere length dynamics were only minimally influenced by manipulation the endogenous telomerase activity in hematopoietic progenitor, despite striking differences in telomerase activity, the exact pathway of telomere length regulation in hematopoietic progenitor is not defined yet; but, it is postulated that the function of telomerase is more tightly regulated in HSCs compared with various other cell types [18]. Mild HT induces differentiation and apoptosis in human leukemic cell lines, and these events were accompanied by reduction in telomerase activity [9, 33]. Yamada et al. [34] showed that down regulation of telomerase activity is an early event of leukemic cells differentiation without apparent telomere DNA changes. Bryan et al. [35] showed that in immortal cells telomere elongation was occurred without detectable telomerase activity. After that many questions, ‘‘such as why telomerase inactivation did not cause telomere shortening in leukemic cells?’’, were raised. Many researches were done to answer to this question in last decade. Immortal tumor cells and cell lines employ a telomere maintenance mechanism that allows them to escape the normal limit on proliferation potential. In leukemic cells, in lower activity of telomerase and absence of telomerase telomere length maybe
689
maintained by an alternative lengthening of telomere (ALT) mechanism [20, 36]. Chare et al. [37] showed that telomere length would be maintained in the absence of telomere associated promyelocytic leukemia bodies containing proleukemia protein, telomeric DNA and telomerebinding proteins too. In similar line to our finding, Pendino et al. reported that retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation too [38]. According to this finding, if telomere length shortage happens due to HT treatment following reduction of telomerase activity in hematopoietic progenitors, then maybe HT can be used as a selective method for treatment of diseases related to control the proliferation of human leukemic and cancer stem cells [39]. And if telomere length shortage would not occur following HT treatment, then stem cell progenitors would not be injured or at least less injured through HT treatment of leukemic cells. Research on telomere and telomerase of stem cells could lead to the development of new treatments for leukemia [40, 41].
Conclusion Our data significantly shows that the telomerase of HSCs are more sensitive to heat treatment than leukemic cells but we did not observe a significant telomere shortening in these cells after treatment by HT at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. In these cells, we did not observed significant telomere shortening in response to mild HT. The mechanism that induces immediate decrease of telomerase activity is undefined. So it seems that the primitive decrease in telomerase activity is related to immediate effect of HT in inactivating enzyme. Acknowledgments This work was supported by educational research funds from National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. The author thanks Ms. Neda VaseliHagh for laboratory technical assistances. Conflict of interest
None.
References 1. Laszlo, A. (1992). The effect of hyperthermia on mammalian cell structure and functions. Cell Proliferation, 25, 59–87. 2. Zhang, Yue, & Calderwood, Stuart K. (2011). Autophagy, protein aggregation and hyperthermia: A mini-review. International Journal of Hyperthermia, 27(5), 409–414. 3. Wada, S., Tabuchi, Y., Kondo, T., Cui, Z. G., Zhao, Q. L., Takasaki, I., et al. (2007). Gene expression in enhanced apoptosis
123
690
4.
5.
6.
7.
8.
9.
10. 11.
12.
13.
14.
15. 16.
17.
18.
19.
20.
21.
Cell Biochem Biophys (2014) 69:681–691 of human lymphoma U937 cells treated with the combination of different free radical generators and hyperthermia. Free Radical Research, 41(1), 73–81. Zintchenko, A., Ogris, M., & Wagner, E. (2006). Temperature dependent gene expression induced by PNIPAM-based copolymers: Potential of hyperthermia in gene transfer. Bioconjugate Chemistry, 17(3), 766–772. Murphy, P. B., & Richman, C. M. (1989). Normal and malignant human myeloid progenitors differ in their sensitivity to hyperthermia. Experimental Hematology, 17, 1105–1109. Setroikromo, R., Wierenga, P. K., van Waarde, M. A., Brunsting, J. F., Vellenga, E., & Kampinga, H. H. (2007). Heat shock proteins and Bcl-2 expression and function in relation to the differential hyperthermic sensitivity between leukemic and normal hematopoietic cells. Cell Stress and Chaperones, 12(4), 320–330. Wierenga, P. K., Setroikromo, R., Kamps, G., Kampinga, H. H., & Vellenga, E. (2003). Differences in heat sensitivity between normal and acute myeloid leukemic stem cells feasibility of hyperthermic purging of leukemic cells from autologous stem cell grafts. Experimental Hematology, 31(5), 421–427. Eppink, Berina, Krawczyk, Przemek M., Stap, Jan, & Kanaar, Roland. (2012). Hyperthermia-induced DNA repair deficiency suggests novel therapeutic anti-cancer strategies. International Journal of Hyperthermia, 28(6), 509–517. Deezagi, A., Manteghi, S., Khosravani, P., Vaseli-Hagh, N., & Soheili, Z. S. (2009). Induced apoptosis by mild hyperthermia occurs via telomerase inhibition on the three human myeloid leukemia cell lines: TF-1, K562 and HL-60. Leukemia and Lymphoma, 50(9), 1519–1527. Collins, K. (2000). Mammalian telomeres and telomerase. Current Opinion in Cell Biology, 12, 378–383. Allsopp, R. C., Chang, E., Kashefi-Aazam, M., et al. (1995). Telomere shortening is associated with cell division in vitro and in vivo. Experimental Cell Research, 220, 194–200. Weinrich, S. L., Pruzan, R., Ma, L., Ouellette, M., Tesmer, V. M., Holt, S. E., et al. (1997). Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nature Genetics, 17, 498–502. Greenwood, Matthew J., & Peter, M. (2003). Lansdorp, telomeres, telomerase, and hematopoietic stem cell biology. Archives of Medical Research, 34, 489–495. Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C. P., Morin, G. B., et al. (1998). Extension of life-span by introduction of telomerase into normal human cells. Science, 279, 349–352. Hoeijmakers, J. H. J. (2001). Genome maintenance mechanisms for preventing cancer. Nature, 411, 366–374. Chiu, C. P., Dragowska, W., Kim, N. W., et al. (1996). Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow. Stem Cells, 14, 239–248. Engelhardt, M., Kumar, R., Albanell, J., et al. (1997). Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood, 90, 182–193. Zimmermann, S., Glaser, S., Ketteler, R., Waller, C. F., Klingmu¨ller, U., & Martensa, U. M. (2004). Effects of telomerase modulation in human hematopoietic progenitor cells. Stem Cells, 22, 741–749. Ohyashiki, K., & Ohyashiki, J. H. (2002). Measurement of telomerase activity in human hematopoietic cells and neoplastic disorders. Methods in Molecular Medicine, 68, 279–299. Weng, N. (2001). Interplay between telomere length and telomerase in human leukocyte differentiation and aging. Journal of Leukocyte Biology, 70, 861–867. Miri-Moghaddam, E., Deezagi, A., & Soheili, Z. S. (2009). Downregulation of telomerase activity in human promyelocytic
123
22.
23.
24.
25.
26.
27.
28.
29. 30.
31.
32.
33.
34.
35.
36.
37.
38.
cell line using RNA interference. Annals of Hematology, 88, 1169–1176. Strunk, D., Rappersberger, K., Egger, C., Strobl, H., Kromer, E., Elbe, A., et al. (1998). Generation of human dendritic cells/langerhans cells from circulating CD34? hematopoietic progenitor cells. Blood, 87(4), 1292–1302. Bartolovic, K., Balabanov, S., Berner, B., Buhring, H. J., Komor, M., Becker, S., et al. (2005). Clonal heterogeneity in growth kinetic of CD34? CD38- human cord blood cells in vitro is correlated with gene expression pattern and telomere length. Stem Cells, 23, 916–957. Deezagi, A., Vaseli-Hagh, N., & Nouri, M. (2011). Studying the anti-aging effect of Nitric Oxide on the cell proliferation and telomerase activity of human cord blood hematopoietic stem cells. International Journal of Hematology, 5(3), 1–10. Keith, W. N., & Monaghan, A. J. (2004). Detection of telomerase activity by TRAP assay. Methods in Molecular Medicine, 97, 311–322. Gammaitoni, L., Weisel, K. C., Gunetti, M., Wu, K. D., Bruno, S., Pinelli, S., et al. (2004). Elevated telomerase activity and minimal telomere loss in cord blood long-term cultures with extensive stem cell replication. Blood, 103(12), 4440–4448. Zhang, W., Piatyszek, M. A., Kobayashi, T., et al. (1996). Telomerase activity in human acute myelogenous leukemia: Inhibition of telomerase activity by differentiation-inducing agents. Clinical Cancer Research, 2, 799–803. Miri-Moghaddam, E., Deezagi, A., Soheili, Z. S., & Shariati, P. (2010). Apoptosis and reduced cell proliferation of HL-60 cell line caused by human telomerase reverse transcriptase inhibition by siRNA. Acta Haematologica, 124, 72–78. Lansdorp, P. M. (2008). Telomeres, stem cells, and hematology. Blood, 111(4), 1759–1765. Agarwal, M., Pandita, S., Hunt, C. R., Gupta, A., Yue, X., Saira Khan, S., et al. (2008). Inhibition of telomerase activity enhances hyperthermia-mediated radiosensitization. Cancer Research, 68(9), 3370–3378. Siddiqui, F., Kolozsvary, A., Barton, K. N., Freytag, S. O., Brown, S. L., & Kim, J. H. (2009). Does hyperthermia increase adenoviral transgene expression or dissemination in tumors? International Journal of Hyperthermia, 25(4), 273–279. Ito, A., Okamoto, N., Yamaguchi, M., Kawabe, Y., & Kamihira, M. (2012). Heat-inducible transgene expression with transcriptional amplification mediated by a transactivator. International Journal of Hyperthermia, 28(8), 788–798. Goliaei, B., & Deizadji, A. (1998). Effects of hyperthermia and granulocyte-macrophage colony stimulating factor on the differentiation of human leukemic cell line U937. Leukemia Research, 22(8), 705–710. Yamada, O., Takanashi, M., Ujihara, M., & Mizoguchi, H. (1998). Down regulation of telomerase activity is an early event of leukemic cells differentiation without apparent telomere DNA change. Leukemia Research, 22(8), 711–717. Bryan, T. M., Englezou, A., Gupta, J., et al. (1995). Telomere elongation in immortal human cells without detectable telomerase activity. EMBO Journal, 14, 4240–4248. Yeager, T. R., Neumann, A. A., Englezou, A., et al. (1999). Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Research, 59, 4175–4179. Fasching, Clare L., Bower, K., & Reddel, R. R. (2005). Telomerase-independent telomere length maintenance in the absence of alternative lengthening of telomeres-associated promyelocytic leukemia bodies. Cancer Research, 65(7), 2722–2729. Pendino, F., Flexor, M., Delhommeau, F., et al. (2001). Retinoids down-regulate telomerase and telomere length in a pathway
Cell Biochem Biophys (2014) 69:681–691 distinct from leukemia cell differentiation. PNAS, 98(12), 6662–6667. 39. Wierenga, P. K., Setroikromo, R., Kamps, G., Kampinga, H. H., & Edo Vellenga, (2002). Peripheral blood stem cells differ from bone marrow stem cells in cell cycle status, repopulating potential, and sensitivity toward hyperthermic purging in mice mobilized with cyclophosphamide and granulocyte colony-stimulating factor. Journal of Hematotherapy & Stem Cell Research, 11(3), 523–532.
691 40. Annaloro, C., Onida, F., Saporiti, G., & Lambertenghi Deliliers, G. (2011). Cancer stem cells in hematological disorders: Current and possible new therapeutic approaches. Current Pharmaceutical Biotechnology, 12(2), 217–225. 41. Rampazzo, E., Bonald, L., Trentin, L., et al. (2012). Telomer length and telomerase levels delineate subgroups of B-cell chronic lymphocytic leukemia with different biological characteristics and clinical outcomes. Haematologica, 97(1), 56–63.
123
ORIGINAL PAPER
Differential Sensitivity of Telomerase from Human Hematopoietic Stem Cells and Leukemic Cell Lines to Mild Hyperthermia Abdolkhaleg Deezagi
Published online: 4 March 2014 Ó Springer Science+Business Media New York 2014
Abstract We have investigated the effects of hyperthermia (HT) on cell proliferation and telomerase activity of human hematopoietic stem cells (HSCs) and compared with human leukemic cell lines (TF-1, K562 and HL-60). The cells were exposed to HT at 42 and 43 °C up to 120 min. The cells were incubated at 37 °C for 96 h. Then the cells were collected and assayed for cell proliferation, viability, telomerase activity, and terminal restriction fragment (TRF) lengths. The enzyme activity from HSCs was decreased up to 68.6 at 42 and 85.1 % at 43 °C for 120 min. This inhibition in leukemic cells was up to 28.9 and 53.6 % in TF-1; 53 and 63.9 % in K562; 45.2 and 61.1 % in HL-60 cells. The treated cells showed TRF lengths about 5.3 kb for control HL-60 cells, 5.0 kb for HL-60 cells treated at 42 and 4.5 kb at 43 °C for 120 min. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0–4.5 kb for treated cells at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. TRF length was constant at treated two types of cells, which implies that in cells subjected to mild HT no telomere shortening was observed. Keywords Hyperthermia Telomerase activity Cell proliferation Hematopoietic stem cells Human leukemic cell lines
A. Deezagi (&) Department of Biochemistry, National Institute of Genetic Engineering and Biotechnology, Km. 17, Karaj-Tehran Freeway, Pajouhesh Blvd., P.O.Box 14155-6343, Tehran, Iran e-mail: [email protected]
Abbreviations HT Hyperthermia TRAP Telomeric repeat amplification protocol assay HSCs Hematopoietic stem cells TRF Terminal restriction fragment
Introduction Heat stress causes a variety of alteration in cellular physiology. One of the well-documented effects of the exposure of mammalian cells to elevated temperatures are the inhibition of macromolecules synthesis, including DNA, RNA and protein synthesis, autophagy, and protein aggregation [1, 2]. Macromolecules, in particular polypeptides, have differential sensitivity to heat stress due to their specific heat capacity [3]. In several humans, hematopoietic and leukemic cell lines exposure to hyperthermia (HT) lead to morphological maturation, repressed cell proliferation and reduced clonally cell growth. These phenotypic changes are linked to change in expression of important growth regulating genes, growth factors or their receptors which finally block the cell division [4]. The human embryo and fetus may be especially vulnerable to chemical and physical insults during defined stages of development. In particular, the scheduled processes of cell proliferation, cell migration, cell differentiation, and apoptosis that occur at different times for different organ structures can be susceptible to elevated temperatures [5]. Existing data on the differential heat sensitivity of normal and malignant hematopoietic cells have encouraged attempts to apply HT as a potential purging agent in the removal of leukemic cells from the donor bone marrow inoculums prior to
123
682
autologous transplantation and novel therapeutic anti-cancer strategies [6–8]. The exact molecular mechanism of the differential sensitivity of leukemic and normal hematopoietic stem cells (HSC) to HT has not been understood. In the previous work, we have showed that the telomerase activity was significantly reduced in three different human leukemic cells (K562, HL-60 and TF-1 cells) in response to HT [9]. Telomeres are the molecular caps at the ends of chromosomes that are composed of repetitive TTAGGG sequences and associated proteins [10]. Telomeres become shorter during somatic development and with the increasing age of the individual [11]. The maintenance of telomeres throughout many cycles of cell division requires the enzyme telomerase, which consists of two core components, the RNA-subunit, hTR, containing the template, and a catalytic protein subunit, human Telomerase Reverse Transcriptase (hTERT) [12]. It has been proposed that telomerase is repressed in somatic tissues, and that telomeres become shorter during somatic development and with the increasing age of the individual [13]. In contrast, leukemic and cancerous cells are characterized by an unlimited proliferative potential, and accumulating evidence suggests that the reactivation of telomerase is a critical step in carcinogenesis and tumorogenesis; also telomerase activity was detected in nearly 90 % of various human tumor samples [14, 15]. Unlike normal somatic cells, telomerase activity has been detected at low levels in hematopoietic progenitor cells, which is up regulated in response to cytokine stimulation [16, 17]. The level of telomerase is low in the majority of human stem cells, whereas it is up regulated in cells that undergo rapid expansion, such as committed hematopoietic progenitor cells, activated lymphocytes, or keratinocytes, even within tissues with a low cell turnover such as the brain [18]. Most cycling HSCs display telomerase activity [19]. Following an initial increase, telomerase activity is down regulated as HSCs, and progenitor cells proliferate and differentiate into more mature cells that display low to negligible levels of telomerase activity [20, 21]. The exact molecular mechanism of the differential sensitivity of leukemic and normal HSCs to HT has not been understood. In this research, we have investigated the telomerase activity and cell proliferation of human HSCs and human leukemic cell lines in response to mild HT (42–43 °C). For this purpose, HSCs were isolated from human cord blood (CB) by immuno-magnetic separation technique. HSCs and human leukemic cell lines were exposed to HT at 42 °C up to 120 min. Then, the cells were incubated at 37 °C for 96 h. Finally, the cells were collected and assayed for cell proliferation, viability, telomerase activity, and telomere length.
123
Cell Biochem Biophys (2014) 69:681–691
Material and Method Human leukemic Cell Culture TF-1 human cell line was purchased from DSMZ Company (DSMZ, Braunschweig, Germany). HL-60 and K562 cells were purchased from American Type Culture Collection (ATCC) (ATCC, Rockville, MD, USA). The Cells were maintained at 37 °C, 5 % CO2 in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10 % FBS (Gibco, Paisley, UK), 120 mg/l penicillin, and 200 mg/l streptomycin. In addition, the culture medium of TF-1 cells was supplemented with 2 ng/ml of rhGM-CSF (Roche, Mannheim, Germany). Cells were cultured at cell density about 2–5 9 105 cells/ml for 4 days. Cell viability was determined by trypan blue-dye exclusion test. Cord Blood Collection Umbilical CB to be discarded were collected. CB was obtained from informed and consenting donors at the Mustafa Khomeini Hospital, Tehran-Iran. The umbilical cord was clamped according to standard hospital procedure, and CB collections were performed ex utero. CB was collected into a sterile collection 50 ml falcon tubes containing 10 ml of Citrate Phosphate Dextrose solution (Sigma, St. Louis, MO, USA). The collection volume varied between 15 and 40 ml. After fourfold dilution with Ca??/Mg?? free Hanks, Balanced Salt Solution (HBSS) (Sigma, St. Louis, MO, USA), mono-nuclear cells (MNCs) were isolated by density gradient using 10 ml of FicollPaque reagent (Nycomed, Oslo, Norway) and 30 ml of diluted CB. The two-phase system was centrifuged at 4009g for 40 min without break, at 20 °C in a swingingbucket rotor (Beckman Coulter Inc., CA, USA). MNCs, collected from the interface of the two phases, were washed twice with PBS. MNCs were counted by hemocytometer neobar, and cells’ viability was assayed by trypan blue dye exclusion test. Separation of CD34?/CD133? Cells Enrichment of HSCs in CB is based on the expression of certain surface antigens, such as CD34 and CD133, or on the lack of expression of lineage-specific antigens. Immunomagnetic negative selection kit (StemSep StemCell Technologies, Vancouver, Canada) was used for the isolation of CD34? or CD133? cells using paramagnetic microbeads conjugated to specific monoclonal antibodies. In negative selection of lineage-negative (Lin-) cells, the unwanted cells are labeled with antibodies against known markers for mature hematopoietic cells (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A)
Cell Biochem Biophys (2014) 69:681–691
and retained in the column. Unlabeled cells pass through the column and are collected as the Lin- cell fraction. To enrich progenitor cells, lineage-committed cells were depleted [22, 23]. The conditions resulting in optimal separation of Lin- cells were 100 ll of Progenitor Enrichment Cocktail and 60 ll of magnetic iron particles per 8 9 107/ml MNCs, as recommended by the manufacturer. MNCs (8 9 107/ml) were labeled with Progenitor Enrichment Cocktail (100 ll/ml) containing antibodies against CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and Glycophorin A (StemCell Technologies, Vancouver, Canada) at room temperature for 15 min. Subsequently, the cell suspension was incubated with magnetic iron particles (60 ll/ml) at room temperature for 15 min. Cell suspension was loaded into MACS LD column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and unlabeled cells passing through the column were collected (Lin- fraction). The column was then washed twice with 1 ml of buffer, and the remaining Lin? cells were collected for control purposes. The isolation method and characterization was carefully described in our last paper [24]. The purity of isolated CD34? HSCs was assayed according the recommendation of the HSC Immunomagnetic negative selection kit (StemSep StemCell Technologies, Vancouver, Canada). Purity was measured using flow cytometery by staining with a FITCconjugated anti-human CD34 antibody catalog number 10413 (StemSep StemCell Technologies, Vancouver, Canada). The cell’s numbers and cell’s viability were determined by a hemocytometer, using the Trypan blue test. The purity of CD34 for suspension culture and various assays, isolated cells were cultured in RPMI-1640 medium (Gibco, Paisley, UK) that supplemented by FCS 20 % with combination of the following cytokines; recombinant human erythropoietin (rhEPO) 6 U/ml (Roche, Mannheim, Germany), recombinant human interleukine-3 (rhIL-3) 50 ng/ml (Sandoze, Basel. Switzerland), recombinant human Granulocyte–Macrophage colony stimulating factor (rhGM-CSF) 20 ng/ml (Sandoze, Basel, Switzerland) and rhIL-6, 20 ng/ml (Sandoze, Basel, Switzerland). Heat Treatment Heat shock was applied at 41–44 °C for different intervals of 15, 30, 60, and 120 min for leukemic cells. Because of limitation in the providing of HCSs, the HSCs were treated only at 42 and 43 °C for 15, 30, 60, and 120 min. The leukemic cells and HSCs (106 cells/ml in RPMI 1640) were separately treated by HT by immersion in thermostated water bath (Memert, Braunschweig, Germany) with ±0.1 °C precision. Control cells were similarly treated at 37 °C. At the end of heat treatment, the cells were allowed to stand for 5 min at room temperature. Cell viability was
683
immediately determined, and 2 9 105 cells were stored for telomerase assay at -20 °C in order to assay the short time effect of HT on the telomerase activity before incubation. Then, the remained portion of the treated cells were transferred to the 6-wells plates (NUNC, Roskilde, Denmark) and cultured in RPMI and 10 % FCS at final cell density of 2 9 105 cells/ml. Cells were incubated at 37 °C for 96 h. Then, the cells were collected and assessed for cell proliferation, telomerase activity, and telomere length as described below. Cell Growth and Proliferation Assays In order to examine the effect of heat shock on the cell growth and cell proliferation, cells were collected after 96 h and the total cell number, viability, and MTT cell proliferation assay were done as below. Cell count and viability were enumerated using a Neobar hemocytometer. MTT Assay After 96 h of incubation, 100 ll of finely resuspended control and heat shock-treated cells were transferred to flat bottom 96 microtiter plates. Then 10 ll of freshly prepared (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, U.S.A.) solution (5 mg/ml in PBS) was added to each well and were incubated for 4 h. Finally, 50 ll of MTT lysis solution (20 % sodium dodecyl sulfate w/v and 50 % dimethyl formamide v/v) was added to each well and incubated overnight. Absorbance was read at 620 nm using an ELISA reader. TRAP-PCR ELISA Assay The telomerase activity was determined using a telomerase repeat amplification protocol TRAP-PCR-ELIZA kit (Roche, Mannheim, Germany) which described by Keith and Monaghan [25] for the first time. This method allows highly specific amplification of telomerase-mediated elongation products combined with nonradioactive detection following an ELISA protocol. The assay separated in two steps. In the first step, telomerase adds telomeric repeats (TTAGGG) to the biotin-labeled synthetic P1TSprimer, and then, the elongation product are amplified using the primers P1-TS. In the second step, PCR product is denatured and hybridized to a digoxigenin-(DIG)— labeled, telomeric repeat-specific detection probe. The resulting product is immobilized via the biotin-labeled primer to a coated microplate. The immobilized PCR product is then detected with an antibody against DIG-POD that is conjugated to peroxidase. Finally, the probe is visualized by virtue of peroxidase metabolizing TMB. Briefly, 2 9 104 cells were washed with cold PBS and
123
684
Cell Biochem Biophys (2014) 69:681–691
lysed in 200 ll of precooled lysis solution and incubated on ice for 30 min, then centrifuged at 16,0009g for 20 min at 4 °C. For PCR amplification, 2 ll of supernatant and 25 ll of reaction mixture (containing Telomerase substrate, primers, nucleotides, and Taq polymerase) were transferred into a suitable tube, and then sterile water was added to a final volume of 50 ll. An amplification reaction was carried out by PCR. The 5 ll amplification product was mixed with 20 ll of denaturation reagent and incubated at room temperature for 10 min, and 225 ll of hybridization buffer was added and mixed thoroughly, then 100 ll of mixture per well was transferred into the precoated microtiter plate and incubated at 37 °C on a shaker for 2 h. Anti-DIC-POD working solution (100 ll) was added and incubated at room temperature for 30 min with shaking. The solution was removed completely, and the precipitate was rinsed five times with 250 ll of washing buffer per well for a minimum of 30 s. After removing the washing buffer, 100 ll of TMB substrate solution was added and incubated for color development at room temperature for 20 min with gentle shaking. Finally, 100 ll of stop reagent was added to each well to stop color development. The amount of TRAP products was determined by the measurement of the absorbance at 450 and 690 nm. A negative as well as a positive control was run each time. A negative control was provided for each extract by heat inactivating the telomerase enzyme present in cell lysate at 65 °C for 10 min prior to the PCR step. Southern Blot Analysis of Telomere Length and Terminal Restriction Fragment (TRF) Length Genomic DNA of the heat-treated HL-60 and HSCs cells was isolated using High Pure Template Preparation kit (Roche, Mannheim, Germany), and TRF length was estimated using Telo TAGGG Telomere Length Assay Kit (Roche, Mannheim, Germany). In brief, 2 lg of genomic DNA was digested with restriction enzymes HinfI and RasI and separated on 0.8 % w/v gel. The DNA fragments were then transferred to a positively charged nylon membrane (Whatman Oxon, UK) in 209 SSC buffer overnight at room temperature. The membrane were hybridized with a digoxigenin (DIG)-labeled telomere-specific probe, and detected by an anti-DIG-alkaline phosphatase and CDPStar as the chemiluminescent substrate. Statistical Analysis Each experiment was performed three times for all data, each carried out in duplicated sequences. Data were analyzed using a one-way analysis of variance (ANOVA) values were given as the mean ± standard deviation (SD), and biological variables were compared using the Students’
123
Fig. 1 Purity of CD34? cells was measured using flow cytometery by staining with a FITC-conjugated anti-human CD34 antibody. The single-parameter histogram and frequency of CD34? cells, a in the started CB, b in the finally enriched and isolated fractions
t test. By convention, a a-level of p \ 0.05 was considered to be statistically significant. Finally, the correlation between heat shock and the telomerase activity, cell proliferation was calculated statistically.
Results HSCs Isolation and Purity Assay CBs were provided from more than 10 newborns. The total cell’s numbers from each step of isolation were counted. Purity of CD34? cells was measured using flow cytometery by staining with a FITC-conjugated anti-human CD34 antibody. The percent of finally isolated total CD34? to started total mononuclear cells was about 1.87 ± 0.61 %. The frequency of CD34? cells in the started CBs were
Cell Biochem Biophys (2014) 69:681–691 1.6
*
95
%of viability
85
*
75
*
65
*
55 45 35
Absorbance at 620 nm
105
685
1.4
*#
1.2
=
1
& 0.8
&
0.6
&
0.4
*= &
0
0
15
30
60
90
120
0
Heat shock time (minutes)
15
30
85
=86#
55
=
#
= =
#
45
#
35
#
25 0
15
30
60
90
120
Heat shock time (minutes) 43 C before incubation
42 C before incubation
43 C after incubation
42 C after incubation
(b) Fig. 2 The effect of heat shock on the cell viability of human eryhtroleukemic TF-1 and human HSCs. The cells were treated by HT (42 and 43 °C up to 120 min), and the cells’ viability was immediately assayed after HT or before incubation. The cells were incubated for 96 h. Then the cells were collected, and the cell viability was detected after incubation. a The TF-1 cells’ viability b human HSCs’ viability. The results are mean ± 1 SEM for four separate experiments, *p \ 0.01 compared untreated versus treated TF-1 cells at 43 °C for 0, 15, 30, 60, 90, and 120 min after 96 h incubation, #p \ 0.01 compared untreated and treated human HSCs cells at 43 °C for 0, 15, 30, 60, 90, and 120 min after 96 h incubation, =p \ 0.05 compared to untreated and treated human HSCs cells at 42 °C for 0, 60, 90, and 120 min after 96 h incubation. In other condition, significant differences were not observed
about 0.9–1.9 % (Fig. 1a) and 51–69 % in the final enriched fractions (Fig. 1b). Cellular Viability and Cell Proliferation After Heat Shock To assess the inhibitory effects of HT on the cell growth, cytotoxic effects of HT on cells was determined using trypan blue exclusion test, cell counting, and MTT cell proliferation assay. The effect of HT on the cell viability was immediately assayed after heat treatment or before incubation and after 96 h incubation. HT caused both
Cell proliferation (% of Controls
95
#
60
120
(a)
(a)
65
45
Heat Shock time (minutes)
105
%of viability
&
0.2
25
75
#
&
120 100 80 60
42 C TF-1 43 C TF-1
40
42 C HSC
20
43 C HSC
0 0
15
30
45
60
120
Heat Shock time (minutes)
(b) Fig. 3 The effect of HT on the cell proliferation of human eryhtroleukemic TF-1 and human HSCs. The cells were treated by HT (42 and 43 °C up to 120 min) and were incubated for 96 h at 37 °C. Then, the cells proliferation was assayed by MTT assay. a Absorbance at 620 nm from MTT assay of the cells. b Percent of cell proliferation of treated cells in comparison to untreated control cells. Values are the means ± 1 SEM of data from duplicate cultures for four separate experiments (n = 4). *p \ 0.01 compared untreated and treated TF-1 cells at 43 °C for 0 and 120 min. =p \ 0.05 compared treated TF-1 cells at 43 °C for 15 and 120 min after 96 h incubation, #p \ 0.01 compared to untreated and treated TF-1 cells at 43 °C for 0 and 60. &p \ 0.01 compared untreated and treated human HSCs cells at 43 °C for 0, 15, 30, 60, 90, and 120 min. In other condition, significant differences were not observed
short- and long-term damages in leukemic cell lines and HSC cells. Short-term damage was expressed as a decrease in cell viability immediately after HT and before incubation. TF-1 cells were viable ([89 %) after 120 min heating at 42 and 43 °C (Fig. 2a). HSCs were sensitive to HT in comparison to TF-1 cells; the viability was decreased in HSCs up to 74.5 ± 3.3 and 62.3 ± 5.9 % at 42 and 43 °C for 120 min HT (Fig. 1b). Long-term damage was expressed when the cells were incubated at 37 °C for 96 h after HT. 120 min of heating at 42 and 43 °C dropped the TF-1 cell viability to 79.8 ± 1.7 and 46.7 ± 10.2 % (Fig. 1a); these condition caused a higher decrease in HSCs viability 64.8 ± 3.8 and 37.6 ± 7.1 %, respectively
123
686
Cell Biochem Biophys (2014) 69:681–691
Fig. 4 Telomerase activity of human leukemic cells and HSCs after treatment by HT. The cells were treated by HT (42 and 43 °C up to 120 min) and telomerase activity was immediately determined after HT using the TRAP-PCR–ELISA method as described in methods. a Telomerase activity after HT at 42 °C (absorbance at 450 nm). b The percent of telomerase activity in comparison to untreated control cells after HT at 42 °C. c Telomerase activity after HT at 43 °C (Absorbance at 450 nm). d The percent of telomerase activity in comparison to untreated control cells after HT at 43 °C. (n = 4), and values are the means ± 1 SE of data from at least two independent experiments. *p \ 0.01 compared untreated and treated
HSCs cells at 42 °C for 0, 15, 30, 60, and 120 min after 96 h incubation. ?p \ 0.01 compared treated leukemic cells at 42 °C for 15, 60, and 120 min after 96 h incubation. #p \ 0.01 compared untreated and treated HSCs cells at 42 °C for 0, 15, 30, 60, and 60 after 96 h incubation. ?p \ 0.01 compared untreated and treated leukemic cells at 43 °C for 0, 15, 30, 60, 90, and 120 min. **p \ 0.01 compared treated leukemic cells at 43 °C for 15 and 120 min. &p \ 0.01 compared treated versus untreated HSCs cells at 43 °C for 0, 15, 30, 60, and 120 min. &&p \ 0.01 compared treated HSCs cells at 43 °C for 15 and 120 min. In other condition, significant differences were not observed
(Fig. 2b). The cell proliferation and growth rate of heat shock-treated cells were assessed using colorimetric MTT cell proliferation assays at each temperature and time points. The cells were treated by HT and cultured (10,000 cell/well in 100 ll in flat bottom 96 wells) at 37 °C for 96 h. Then MTT dye was added, and absorbance was measured. The values for two types of cells were averaged, and growth curves were constructed. The values for untreated control cells were 1.18 ± 0.24 absorbance unit (AU) for TF-1 cells and 0.72 ± 0.12 AU for HSCs (Fig. 3a). The results of the MTT cell proliferation assay indicated to the low potency of HSCs in comparison to TF1 cells. Results of the HT-treated cells indicated that the cells proliferation was significantly decreased with the increasing of time and temperature in both type of cells. When the cells were treated by HT up to 120 min, the cell
proliferation potency was decrease up to 40 % (0.433 ± 0.048 AU) for HSCs and up to 30 % (0.824 ± 0.171 AU) for TF-1 cells in comparison to untreated control cells at 42 °C for 120 min. Treatment of cells by HT at 43 °C caused a sharp reduction of cell proliferation up to 71 % (0.211 ± 0.02 AU) for HSCs and 52 % (0.56 ± 0.12 AU) for TF-1 cells (Fig. 3b).
123
Effect of HT on Telomerase Activity To examine the effect of heat shock on telomerase activity, the activity of enzyme was assayed by TRAP-PCR ELISA method as described in methods. The telomerase activity was analyzed immediately and over 96 h incubation after heat shock. When the cells were treated by HT and telomerase activity was assayed before incubation, the enzyme
Cell Biochem Biophys (2014) 69:681–691
687
Fig. 5 Telomerase activity of human leukemic cells and HSCs after treatment by HT and incubation for 96 h. The cells were treated by HT (42 and 43 °C up to 120 min) and were incubated for 96 h at 37 °C. Telomerase activity was determined using the TRAP-PCR– ELISA method as described in methods. a Telomerase activity after HT at 42 °C (absorbance at 450 nm). b The percent of telomerase activity in comparison to untreated control cells after HT at 42 °C. c Telomerase activity after HT at 43 °C (absorbance at 450 nm). d The percent of telomerase activity in comparison to untreated control cells after HT at 43 °C. (n = 4), and values are the
means ± 1 SE of data from at least two independent experiments. *p \ 0.01 compared to untreated versus treated leukemic cells at 42 °C for 0, 30, 60, and 120 min. #p \ 0.01 compared to treated HSCs cells at 43 °C for 0, 15, 30, 60, and 120 min after 96 h incubation. ##p \ 0.01 compared to untreated and treated leukemic cells at 43 °C for 0, 15, 30, 60, and 120 min after 96 h incubation. **p \ 0.01 compared to treated and untreated HSCs cells at 43 °C for 0, 15, 30, 60, and 120 min after 96 h incubation. In other condition, significant differences were not observed
activity from HSCs was reduced up to 58 % at 42 °C for 120 min (0.260 ± 0.018 AU for control cells, 0.109 ± 0.016 at 42 °C for 120 min). This reduction in leukemic cells was about 47.3 % in TF-1 cells (0.701 ± 0.002 and 0.370 ± 0.057 AU), 30.5 % in K562 cells (0.685 ± 0.042 and 0.544 ± 0.029 AU), and 31.4 % in HL-60 cells (0.617 ± 0.064 for control cells and 0.426 ± 0.082 AU for treated cells at 42 °C for 120 min), respectively, Fig. 4a, b. The enzyme activity from HSCs was reduced up to 73.1 % at 43 °C for 120 min (0.260 ± 0.018 AU for control cells and 0.072 ± 0.009 AU at 43 °C for 120 min). This reduction in leukemic cells was about 52.2 % in TF-1 cells (0.701 ± 0.002 and 0.355 ± 0.011 AU), 35.9 % in K562 cells (0.679 ± 0.021 and 0.435 ± 0.028 AU), and 37.7 % in HL-60 cells (0.617 ± 0.06 4 for untreated control cells and 0.405 ± 0.058 AU for treated cells at 43 °C for 120 min), respectively, Fig. 4c, d.
When the cells were incubated 96 h at 37 °C after HT, the telomerase activity of HSCs was significantly reduced in comparison to leukemic cells. The enzyme activity from HSCs was decreased up to 68.6 at 42 °C for 120 min (0.24 ± 0.014 for control cells and 0.076 ± 0.024 AU at 42 °C for 120 min). This inhibition in leukemic cells was up to 28.9 in TF-1 cell (0.780 ± 0.058 and 0.573 ± 0.036 AU), 53 % in K562 cells (1.126 ± 0.107 and 0.529 ± 0.15 AU), and 45.2 % in HL-60 cells (0.991 ± 0.024 for untreated control cells and 0.543 ± 0.036 AU for treated cells at 42 °C for 120 min), respectively at that conditions (Fig. 5a, b). The enzyme activity from HSCs was decreased up to 85.1 % at 43 °C for 120 min (0.24 ± 0.014 for control cells and 0.035 ± 0.005 AU at 43 °C). This inhibition in leukemic cells was up to 53.6 % in TF-1 cells (0.780 ± 0.058 and 0.35 ± 0.033 AU), 63.9 % in K562 cells (1.124 ± 0.126 and 0.405 ± 0.074 AU), and 61.1 % in HL-60 cells (0.897 ± 0.039 and
123
688
Fig. 6 Southern blot analysis of the effects of HT on telomere length. The HL-60 and HSCs cells were exposed to HT at 42 and 43 °C for 120 min. Then, the cells were incubated at 37 °C for 96 h, and genomic DNA was extracted and analyzed as described in methods for telomere length. The TRF length of DNA from the treated and control cells were determined. MW molecular weight DNA Marker, P positive control, N negative control, lane 1 untreated HL-60 cells; HL-60 cells treated by HT at 42 °C (lane 2) and 43 °C (lane 3) for 120 min; lane 4 untreated HSCs cells; HSCs cells treated by HT at 42 °C (lane 5) and 43 °C (lane 6) for 120 min
0.351 ± 0.034 AU), respectively at that conditions (Fig. 5c, d). The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. Effect of Heat Treatment on Telomere Length and TRF Length Southern blot analysis was carried out after treatment of cells at 42 and 43 °C for 120 min and 96 h incubation at 37 °C. Figure 6 reveals that the pattern of TRF length did not show a significant difference in heat-treated HL-60 cells at 42 and 43 °C in comparison with the untreated control cells (lanes 1, 2, 3). The treated cells showed that the TRF lengths of untreated HL-60 cells ranged approximately from 3.2 to 7.4 kb, with a mean value of 5.3 kb, averaged 5.0 kb for 120 min at 42 °C and 4.5 kb for 120 min at 43 °C. These results were constant at treated HL-60 cells which implies that in cells subjected to HT at 42 and 43 °C for 120 min no telomere shortening was observed. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0–4.5 kb for treated cells at 42 and 43 °C for 120 min (lanes 4, 5 and 6). In this cell, we did not observed significant telomere shortening too.
Discussion Here, we analyzed short-term effects of mild HT to evaluate telomerase activity and the rate of sensitivity of
123
Cell Biochem Biophys (2014) 69:681–691
hematopoietic cells. According the previous reported data about the high sensitivity of leukemic cells to HT, our cell survival and cell proliferation data indicated to the hyperthermic sensitivity of leukemic cells in comparison to HSCs too. In spite of hyperthermic sensitivity of leukemic cells, we provide evidence that the telomerase activity of HSCs is more sensitive to HT than leukemic cell lines. The mechanism that induces immediate decrease of telomerase activity did not understand. So it seems that the primitive decrease in telomerase activity is related to immediate effect of HT in inactivating enzyme [9]. The presence of telomerase activity in hematopoietic stem/progenitor cells in comparison to the majority of human somatic cells implies an important function. While telomerase expression may serve to delay telomere shortening in these cells, telomere attrition is not prevented. It is beginning to emerge that telomerase may play an additional role to telomere maintenance in hematopoietic cell function [26]. The expanding development of specific telomerase inhibitors for the treatment of leukemia and cancer by induction of apoptosis, including hematopoietic malignancies, necessitates the urgent clarification of telomerase function in hematopoietic cells and malignancies [27, 28]. It is crucial that the role of telomerase in both normal hematopoiesis and leukemia is understood, allowing us to determine the potential effects of clinical inhibition or activation of telomerase on HSC function [29]. Agrawal et al. previously presented that moderate HT (43 °C) enhances telomerase activity. Inhibition of telomerase activity with human telomerase RNA-targeted antisense agents, and in particular GRN163L, results in enhanced HT-mediated IR-induced cell killing, and ectopic expression of catalytic unit of telomerase (TERT) [30]. Our findings were not in agreement with their results. For explanation and clear discussion, it must be considered that the type of the cells used by us is different from that of theirs. Agrawal et al. used 293, HeLa, GM857, and primary fibroblast cell lines (GM5823, HFF, and BJ) with their corresponding stably transfected hTERT counterparts (GM5823?hTERT, BJ?hTERT, and HFF?hTERT) Tercnull and Tert-null mouse embryonic cells along with their wild-type counterparts. They used multiple approaches to determine the effect of telomerase inactivation by heat. They concluded interestingly, heat does not induce any telomerase activity in primary fibroblasts (BJ or GM5823) and, when treated with telomerase inhibitors, did not show any major influence on clonogenic survival after heat shock and IR treatments compared with the telomerase-positive cells [30]. Therefore, their finding in primary cells and wild type cancerous cells indicated to the inhibition of telomerase activity by HT at 43 °C for 1 h. But in over expression systems with transfection of these cells by hTERT, they showed telomerase activity was increased. For clear
Cell Biochem Biophys (2014) 69:681–691
discussion of telomerase activation by HT in over expression systems, it must consider that, viral vectors and transfection systems used for cancer gene therapy are usually delivered by direct intratumoral administration, and HT can affect the transfection and expression efficiency in those systems. Siddiqui et al. [31] studied the role of HT in vitro and in vivo in an attempt to achieve higher transfection rates (especially, larger volume of spread). They concluded that, in the in vitro studies, A549 cells infected with the adenoviral construct did not show any difference in gene expression level in the presence or absence of HT. In vivo, the effect of HT on the volume of gene expression in A549 tumors was highly variable with some groups of mice showing better spread in the presence of HT and others showing reduced spread with HT [31]. Ito et al. recently reported that heat-inducible transgene expression with transcriptional amplification mediated by a transactivator. They have combined heat inducibility with highlevel transgene expression, developed a heat-inducible transgene expression system with transcriptional amplification mediated by a tetracycline-responsive transactivator [32]. Zintchenko et al. [4] have shown the temperature dependency of gene expression which induced by PNIPAM-based copolymers and the potential of HT in gene transfer too. Our data significantly show that the telomerase of HSCs are more sensitive to heat treatment than malignant hematopoietic and leukemic cells, but we did not observe a significant telomere shortening in these cells after treatment by HT at 42 and 43 °C for 120 min. In one study, telomere length dynamics were only minimally influenced by manipulation the endogenous telomerase activity in hematopoietic progenitor, despite striking differences in telomerase activity, the exact pathway of telomere length regulation in hematopoietic progenitor is not defined yet; but, it is postulated that the function of telomerase is more tightly regulated in HSCs compared with various other cell types [18]. Mild HT induces differentiation and apoptosis in human leukemic cell lines, and these events were accompanied by reduction in telomerase activity [9, 33]. Yamada et al. [34] showed that down regulation of telomerase activity is an early event of leukemic cells differentiation without apparent telomere DNA changes. Bryan et al. [35] showed that in immortal cells telomere elongation was occurred without detectable telomerase activity. After that many questions, ‘‘such as why telomerase inactivation did not cause telomere shortening in leukemic cells?’’, were raised. Many researches were done to answer to this question in last decade. Immortal tumor cells and cell lines employ a telomere maintenance mechanism that allows them to escape the normal limit on proliferation potential. In leukemic cells, in lower activity of telomerase and absence of telomerase telomere length maybe
689
maintained by an alternative lengthening of telomere (ALT) mechanism [20, 36]. Chare et al. [37] showed that telomere length would be maintained in the absence of telomere associated promyelocytic leukemia bodies containing proleukemia protein, telomeric DNA and telomerebinding proteins too. In similar line to our finding, Pendino et al. reported that retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation too [38]. According to this finding, if telomere length shortage happens due to HT treatment following reduction of telomerase activity in hematopoietic progenitors, then maybe HT can be used as a selective method for treatment of diseases related to control the proliferation of human leukemic and cancer stem cells [39]. And if telomere length shortage would not occur following HT treatment, then stem cell progenitors would not be injured or at least less injured through HT treatment of leukemic cells. Research on telomere and telomerase of stem cells could lead to the development of new treatments for leukemia [40, 41].
Conclusion Our data significantly shows that the telomerase of HSCs are more sensitive to heat treatment than leukemic cells but we did not observe a significant telomere shortening in these cells after treatment by HT at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. In these cells, we did not observed significant telomere shortening in response to mild HT. The mechanism that induces immediate decrease of telomerase activity is undefined. So it seems that the primitive decrease in telomerase activity is related to immediate effect of HT in inactivating enzyme. Acknowledgments This work was supported by educational research funds from National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. The author thanks Ms. Neda VaseliHagh for laboratory technical assistances. Conflict of interest
None.
References 1. Laszlo, A. (1992). The effect of hyperthermia on mammalian cell structure and functions. Cell Proliferation, 25, 59–87. 2. Zhang, Yue, & Calderwood, Stuart K. (2011). Autophagy, protein aggregation and hyperthermia: A mini-review. International Journal of Hyperthermia, 27(5), 409–414. 3. Wada, S., Tabuchi, Y., Kondo, T., Cui, Z. G., Zhao, Q. L., Takasaki, I., et al. (2007). Gene expression in enhanced apoptosis
123
690
4.
5.
6.
7.
8.
9.
10. 11.
12.
13.
14.
15. 16.
17.
18.
19.
20.
21.
Cell Biochem Biophys (2014) 69:681–691 of human lymphoma U937 cells treated with the combination of different free radical generators and hyperthermia. Free Radical Research, 41(1), 73–81. Zintchenko, A., Ogris, M., & Wagner, E. (2006). Temperature dependent gene expression induced by PNIPAM-based copolymers: Potential of hyperthermia in gene transfer. Bioconjugate Chemistry, 17(3), 766–772. Murphy, P. B., & Richman, C. M. (1989). Normal and malignant human myeloid progenitors differ in their sensitivity to hyperthermia. Experimental Hematology, 17, 1105–1109. Setroikromo, R., Wierenga, P. K., van Waarde, M. A., Brunsting, J. F., Vellenga, E., & Kampinga, H. H. (2007). Heat shock proteins and Bcl-2 expression and function in relation to the differential hyperthermic sensitivity between leukemic and normal hematopoietic cells. Cell Stress and Chaperones, 12(4), 320–330. Wierenga, P. K., Setroikromo, R., Kamps, G., Kampinga, H. H., & Vellenga, E. (2003). Differences in heat sensitivity between normal and acute myeloid leukemic stem cells feasibility of hyperthermic purging of leukemic cells from autologous stem cell grafts. Experimental Hematology, 31(5), 421–427. Eppink, Berina, Krawczyk, Przemek M., Stap, Jan, & Kanaar, Roland. (2012). Hyperthermia-induced DNA repair deficiency suggests novel therapeutic anti-cancer strategies. International Journal of Hyperthermia, 28(6), 509–517. Deezagi, A., Manteghi, S., Khosravani, P., Vaseli-Hagh, N., & Soheili, Z. S. (2009). Induced apoptosis by mild hyperthermia occurs via telomerase inhibition on the three human myeloid leukemia cell lines: TF-1, K562 and HL-60. Leukemia and Lymphoma, 50(9), 1519–1527. Collins, K. (2000). Mammalian telomeres and telomerase. Current Opinion in Cell Biology, 12, 378–383. Allsopp, R. C., Chang, E., Kashefi-Aazam, M., et al. (1995). Telomere shortening is associated with cell division in vitro and in vivo. Experimental Cell Research, 220, 194–200. Weinrich, S. L., Pruzan, R., Ma, L., Ouellette, M., Tesmer, V. M., Holt, S. E., et al. (1997). Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nature Genetics, 17, 498–502. Greenwood, Matthew J., & Peter, M. (2003). Lansdorp, telomeres, telomerase, and hematopoietic stem cell biology. Archives of Medical Research, 34, 489–495. Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C. P., Morin, G. B., et al. (1998). Extension of life-span by introduction of telomerase into normal human cells. Science, 279, 349–352. Hoeijmakers, J. H. J. (2001). Genome maintenance mechanisms for preventing cancer. Nature, 411, 366–374. Chiu, C. P., Dragowska, W., Kim, N. W., et al. (1996). Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow. Stem Cells, 14, 239–248. Engelhardt, M., Kumar, R., Albanell, J., et al. (1997). Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood, 90, 182–193. Zimmermann, S., Glaser, S., Ketteler, R., Waller, C. F., Klingmu¨ller, U., & Martensa, U. M. (2004). Effects of telomerase modulation in human hematopoietic progenitor cells. Stem Cells, 22, 741–749. Ohyashiki, K., & Ohyashiki, J. H. (2002). Measurement of telomerase activity in human hematopoietic cells and neoplastic disorders. Methods in Molecular Medicine, 68, 279–299. Weng, N. (2001). Interplay between telomere length and telomerase in human leukocyte differentiation and aging. Journal of Leukocyte Biology, 70, 861–867. Miri-Moghaddam, E., Deezagi, A., & Soheili, Z. S. (2009). Downregulation of telomerase activity in human promyelocytic
123
22.
23.
24.
25.
26.
27.
28.
29. 30.
31.
32.
33.
34.
35.
36.
37.
38.
cell line using RNA interference. Annals of Hematology, 88, 1169–1176. Strunk, D., Rappersberger, K., Egger, C., Strobl, H., Kromer, E., Elbe, A., et al. (1998). Generation of human dendritic cells/langerhans cells from circulating CD34? hematopoietic progenitor cells. Blood, 87(4), 1292–1302. Bartolovic, K., Balabanov, S., Berner, B., Buhring, H. J., Komor, M., Becker, S., et al. (2005). Clonal heterogeneity in growth kinetic of CD34? CD38- human cord blood cells in vitro is correlated with gene expression pattern and telomere length. Stem Cells, 23, 916–957. Deezagi, A., Vaseli-Hagh, N., & Nouri, M. (2011). Studying the anti-aging effect of Nitric Oxide on the cell proliferation and telomerase activity of human cord blood hematopoietic stem cells. International Journal of Hematology, 5(3), 1–10. Keith, W. N., & Monaghan, A. J. (2004). Detection of telomerase activity by TRAP assay. Methods in Molecular Medicine, 97, 311–322. Gammaitoni, L., Weisel, K. C., Gunetti, M., Wu, K. D., Bruno, S., Pinelli, S., et al. (2004). Elevated telomerase activity and minimal telomere loss in cord blood long-term cultures with extensive stem cell replication. Blood, 103(12), 4440–4448. Zhang, W., Piatyszek, M. A., Kobayashi, T., et al. (1996). Telomerase activity in human acute myelogenous leukemia: Inhibition of telomerase activity by differentiation-inducing agents. Clinical Cancer Research, 2, 799–803. Miri-Moghaddam, E., Deezagi, A., Soheili, Z. S., & Shariati, P. (2010). Apoptosis and reduced cell proliferation of HL-60 cell line caused by human telomerase reverse transcriptase inhibition by siRNA. Acta Haematologica, 124, 72–78. Lansdorp, P. M. (2008). Telomeres, stem cells, and hematology. Blood, 111(4), 1759–1765. Agarwal, M., Pandita, S., Hunt, C. R., Gupta, A., Yue, X., Saira Khan, S., et al. (2008). Inhibition of telomerase activity enhances hyperthermia-mediated radiosensitization. Cancer Research, 68(9), 3370–3378. Siddiqui, F., Kolozsvary, A., Barton, K. N., Freytag, S. O., Brown, S. L., & Kim, J. H. (2009). Does hyperthermia increase adenoviral transgene expression or dissemination in tumors? International Journal of Hyperthermia, 25(4), 273–279. Ito, A., Okamoto, N., Yamaguchi, M., Kawabe, Y., & Kamihira, M. (2012). Heat-inducible transgene expression with transcriptional amplification mediated by a transactivator. International Journal of Hyperthermia, 28(8), 788–798. Goliaei, B., & Deizadji, A. (1998). Effects of hyperthermia and granulocyte-macrophage colony stimulating factor on the differentiation of human leukemic cell line U937. Leukemia Research, 22(8), 705–710. Yamada, O., Takanashi, M., Ujihara, M., & Mizoguchi, H. (1998). Down regulation of telomerase activity is an early event of leukemic cells differentiation without apparent telomere DNA change. Leukemia Research, 22(8), 711–717. Bryan, T. M., Englezou, A., Gupta, J., et al. (1995). Telomere elongation in immortal human cells without detectable telomerase activity. EMBO Journal, 14, 4240–4248. Yeager, T. R., Neumann, A. A., Englezou, A., et al. (1999). Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Research, 59, 4175–4179. Fasching, Clare L., Bower, K., & Reddel, R. R. (2005). Telomerase-independent telomere length maintenance in the absence of alternative lengthening of telomeres-associated promyelocytic leukemia bodies. Cancer Research, 65(7), 2722–2729. Pendino, F., Flexor, M., Delhommeau, F., et al. (2001). Retinoids down-regulate telomerase and telomere length in a pathway
Cell Biochem Biophys (2014) 69:681–691 distinct from leukemia cell differentiation. PNAS, 98(12), 6662–6667. 39. Wierenga, P. K., Setroikromo, R., Kamps, G., Kampinga, H. H., & Edo Vellenga, (2002). Peripheral blood stem cells differ from bone marrow stem cells in cell cycle status, repopulating potential, and sensitivity toward hyperthermic purging in mice mobilized with cyclophosphamide and granulocyte colony-stimulating factor. Journal of Hematotherapy & Stem Cell Research, 11(3), 523–532.
691 40. Annaloro, C., Onida, F., Saporiti, G., & Lambertenghi Deliliers, G. (2011). Cancer stem cells in hematological disorders: Current and possible new therapeutic approaches. Current Pharmaceutical Biotechnology, 12(2), 217–225. 41. Rampazzo, E., Bonald, L., Trentin, L., et al. (2012). Telomer length and telomerase levels delineate subgroups of B-cell chronic lymphocytic leukemia with different biological characteristics and clinical outcomes. Haematologica, 97(1), 56–63.
123
