Gene 562 (2015) 145–151
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Augmented telomerase activity, reduced telomere length and the presence of alternative lengthening of telomere in renal cell carcinoma: Plausible predictive and diagnostic markers Deeksha Pal a, Ujjawal Sharma a, Ragini Khajuria a, Shrawan Kumar Singh b, Nandita Kakkar c, Rajendra Prasad a,⁎ a b c
Department of Biochemistry, PGIMER, Chandigarh, India Department of Urology, PGIMER, Chandigarh, India Department of Histopathology, PGIMER, Chandigarh, India
a r t i c l e
i n f o
Article history: Received 18 November 2014 Received in revised form 4 February 2015 Accepted 12 February 2015 Available online 11 March 2015 Keywords: Telomerase Telomere length PML bodies ALT TRAP
a b s t r a c t In this study, we analyzed 100 cases of renal cell carcinoma (RCC) for telomerase activity, telomere length and alternative lengthening of telomeres (ALT) using the TRAP assay, TeloTTAGGG assay kit and immunohistochemical analysis of ALT associated promyelocytic leukemia (PML) bodies respectively. A significantly higher (P = 0.000) telomerase activity was observed in 81 cases of RCC which was correlated with clinicopathological features of tumor for instance, stage (P = 0.008) and grades (P = 0.000) but not with the subtypes of RCC (P = 0.355). Notwithstanding, no correlation was found between telomerase activity and subtypes of RCC. Strikingly, the telomere length was found to be significantly shorter in RCC (P = 0.000) to that of corresponding normal renal tissues and it is well correlated with grades (P = 0.016) but not with stages (P = 0.202) and subtypes (P = 0.669) of RCC. In this study, telomere length was also negatively correlated with the age of patients (r2 = 0.528; P = 0.000) which supports the notion that it could be used as a marker for biological aging. ALT associated PML bodies containing PML protein was found in telomerase negative cases of RCC. It suggests the presence of an ALT pathway mechanism to maintain the telomere length in telomerase negative RCC tissues which was associated with high stages of RCC, suggesting a prevalent mechanism for telomere maintenance in high stages. In conclusion, the telomerase activity and telomere length can be used as a diagnostic as well as a predictive marker in RCC. The prevalence of ALT mechanism in high stages of RCC is warranted for the development of anti-ALT inhibitors along with telomerase inhibitor against RCC as a therapeutic approach. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Renal cell carcinoma (RCC) is the most prevalent and most lethal cancer of the kidney which accounts for approximately 3% of adult malignancies (Pal et al., 2014). A substantial gain in its incidence has been reported during the last decades (Siegel et al., 2014). Histopathologically, about 80% of RCCs are the clear cell type and the rest of the RCC subtypes include papillary, chromophobe & collecting duct. The clinical diagnosis of RCC is often confirmed by imaging studies, including Xray and computed tomography but the possible existence of benign renal tumor is a serious challenge to the diagnosis. Importantly, RCC is one of the most therapy resistant cancers. It responds very poorly or not at all to chemotherapy and hormonal and radiation therapies
Abbreviations: RCC, renal cell carcinoma; ALT, alternative lengthening of telomeres; PML, promyelocytic leukemia; TRAP, telomere repeat amplification protocol. ⁎ Corresponding author. E-mail address: [email protected] (R. Prasad).
http://dx.doi.org/10.1016/j.gene.2015.02.079 0378-1119/© 2015 Elsevier B.V. All rights reserved.
(Walsh et al., 2002). It is notable that a better understanding of RCC tumor biology at the molecular level is utmost important to improve current diagnosis, prognosis and treatment of RCC. One of the hallmarks of cancer is unlimited proliferation capacity, which is strictly associated with the ability to maintain telomeres through the activation of telomerase (Xu et al., 2013). Telomeres consist of 4 to 15 kb of the repetitive hexamer DNA sequence (TTAGGG) at the ends of chromosomes and undergo progressive shortening approximately 50–100 bp with each round of cell division (Blackburn, 1991). The repetitive noncoding telomeric repeats function as a buffer zone preventing the adjacent coding region of the genome from erosion by the telomeric DNA shortening. Telomerase, or telomere terminal transferase, is a ribonucleoprotein that involves in the de novo synthesis and elongation of telomeric repeats onto chromosomal ends by adding (TTAGGG)n repeats (Blackburn, 1991). Functionally, immortal germ cell lines express telomerase and maintain sufficient telomeric repeats, whereas most human somatic cells are unable to acquire telomerase activity in successive cell culture and become senescent (Allsopp et al., 1995).
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Stabilization of telomeres by telomerase reactivation appears to be concomitant with attainment of immortality which is likely to be necessary for infinite tumor growth. Most of the cancer cells maintain the telomere length with telomerase (Kim et al., 1994). Fewer yet significant numbers of tumor cells bypass the end replication in a telomerase independent manner, the process of which is called alternative lengthening of telomeres (ALT). Several lines of evidences indicate that an ALT involves a recombination based mechanism (Dunham et al., 2000) and characteristics of ALT cells include long and heterogeneous telomeres and subnuclear structure, termed as ALT associated promyelocyctic leukemia (PML) bodies (APBs). These structures contain telomeric DNA, telomere specific binding proteins (TRF1 & TRF2) and proteins involved in DNA replication and recombination (Cesare and Reddel, 2010). However, in few cases of cancers telomere maintenance mechanism is still not known (Ulaner et al., 2003). There are mounting evidences for the existence of a significant relationship between telomeres, telomerase and cancer and cancer mortality (Artandi and DePinho, 2010; Willeit et al., 2010; Shay and Wright, 2011). There are only few reports available in the literature on evaluation of telomerase activity (Yoshida et al., 1998; Fujioka et al., 2000) as well as telomere length in RCC (Mehle et al., 1994; Dahse et al., 1999). Approximately, 85% of all human tumors have telomerase activity (Hrdlicková et al., 2012) and specific cancer subset exhibits the ALT phenotype, a telomerase independent telomere maintenance mechanism (Nguyen et al., 2013). Nevertheless, the presence of ALT in human RCC has not been studied extensively. Moreover, assessments of telomerase activity, telomere length and ALT as well as their association with grades and stages have not been evaluated in RCC patients. In view of these facts, a comprehensive study was extended to interpret the status of telomerase activity, telomere length and ALT in RCC progression, diagnosis and the future treatment by targeting telomerase/telomere or ALT pathway. 2. Materials and methods
total protein extract for 20 min at 85 °C. The ELISA was performed in triplicate. The relative telomerase activities (RTA) of different samples were calculated byðAS−AS; OÞ=AS; IS x100 ðATS8−ATS8; 0Þ=ATS8; IS
RTA ¼ where
AS AS,0 AS,IS ATS8 ATS8,0 ATS8,IS
absorbance of the sample absorbance of heat treated samples absorbance of internal standard of the sample (IS) absorbance of control template (TS80) absorbance of lysis buffer absorbance of internal standard (IS) of the control template (TS8).
2.3. Telomere restriction fragment length analysis The genomic DNA was extracted using the standard method, with proteinase K treatment and phenol/chloroform extraction from 25 to 50 mg of the frozen tissue (Ausubel et al., 2002). Telomere length was assessed by measuring the telomere restriction fragments using the TeloTTAGGG telomere length assay kit (Roche Diagnostics, Indianapolis, USA) by following manufacturer's instructions. After digestion of the genomic DNA with Hinf1 and Rsa1 restriction enzymes, fragments were separated on a 0.8% agarose gel. The DNA was then transferred onto the nylon membrane (Roche Diagnostics, Indianpolis, USA) by Southern blotting and the blotted DNA fragments were hybridized with digoxigeninlabeled probe specific for telomeric repeats and incubated with a digoxigenin-specific antibody covalently coupled to alkaline phosphate. Finally, the immobilized telomere probe was visualized by metabolizing CDP-Star, a highly sensitive chemiluminescence substrate. The average TRF length was calculated by the following formula given in the kit.
2.1. Tissue specimens The present study was approved by the institute ethics committee and informed consent was obtained from patients. Following nephrectomy, tissue samples were taken from the tumor and grossly normal renal parenchyma separately. The samples were snap frozen in liquid nitrogen and stored at −80 °C till further use. Grading of tumors was done by the Fuhrman grading system (Fuhrman et al., 1982). 2.2. Telomerase activity by telomere repeat amplification protocol (TRAP) assay The tissue samples (50–100 mg) were homogenized and grinded after freezing in liquid nitrogen. The finely minced tissue was then extracted with lysis solution from the TeloTAGGG Telomerase PCR ELISAPLUS Kit (Roche Diagnostics, Indianapolis, USA) following the manufacturer's recommendations. The supernatant was transferred to a fresh tube and the total protein concentration was determined by the Lowry method (Lowry et al., 1951). Subsequently, TRAP reaction was carried out with 5 μg of total protein extract in a final volume of 50 μl, starting with the primer elongation step for 30 min at 25 °C, followed by telomerase inactivation for 5 min at 94 °C and 33 PCR cycles with denaturing for 30 s (94 °C), annealing for 30 s (50 °C), and polymerization for 90 s (72 °C). A 5 μl aliquot of the PCR product was denatured, hybridized to a DIG-labeled telomeric repeat-specific probe and immobilized onto a streptavidin-coated microtiter plate via the biotin-labeled synthetic PlTS primer used for PCR. The amount of immobilized PCR product was determined with a peroxidase-conjugated anti-DIG antibody followed by a 3,3′,5,5'-tetramethylbenzidine (TMB) substrate color reaction. Absorbance was measured at 450 nm (reference wavelength, 650 nm) using an ELISA reader. Negative controls were prepared by incubating the
TRF ¼
∑ðODi Þ ∑ðODi =Li Þ
where, ODi Li
chemiluminescent signal length of TRF at position i.
2.4. Immunohistochemical analysis of ALT associated PML bodies Goat polyclonal IgG antibody directed against the N-terminus of the human PML protein (Santa Cruz, USA) was used for immunostaining at 1:100 dilutions (Sharma et al., 2014). Briefly, 5 μm tissue sections were cut, deparaffinized in xylene followed by rehydration in 100%, 95% and 70% ethanol. Antigen retrieval was carried out by boiling the slides in 10 mM citrate buffer, pH 6.0 for 15 min. Slides were then treated with blocking serum for 10 min, after which they were incubated with primary antibody at 4 °C overnight in a humid chamber, followed by incubation with HRP anti-goat IgG (1:100) secondary antibody overnight in a humid chamber (Bangalore Geni, India). Chromogen detection was performed with diaminobenzidine (DAKO Corp., Carpinteria, CA) solution (0.5 ml of stock DAB in 4.5 ml of Tris buffer with 20 μl of hydrogen peroxide). Slides were counterstained with hematoxylin and photographed. 2.5. Statistical analysis Statistical evaluation was performed using SPSS program (version 20.0; SPSS Inc., Chicago, IL). Differences in mean telomerase activity as well as relative telomere length between two groups were evaluated using Mann–Whitney and between more than two groups were
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evaluated using Kruskal–Wallis test. To analyze correlation between tumor grades, stages, subtypes of RCC and telomerase activity, telomere length Spearman rank correlation test was used. The correlation of PML positivity and high stages of RCC was evaluated by Pearson Chi-Square test. A P-value of b0.05 was considered significant. 3. Results 3.1. Patient's characteristics A total of 100 cases of histopathologically proven RCC were included in this study that includes 69 cases of clear cell carcinoma, 13 papillary, 7 sarcomatoid, 6 chromophobe, 4 oncocytoma and 1 of collecting duct carcinoma. Detailed patient's characteristics are given in Table 1. 3.2. Telomerase activity and its correlation with histological characteristics TRAP assay revealed detectable telomerase activity in 81% of the cases of RCC and it was found to be significantly higher to that of adjacent normal renal parenchyma (P = 0.000; Fig. 1A). Telomerase activity was present in 54 of 69 clear cell RCCs (78.3%), 10 of 13 cases of papillary RCCs (76.9%), 6 of 7 (85.7%) cases of sarcomatoid RCCs, 6 of 6 (100%) cases of chromophobe RCCs, 4 of 4 (100%) cases of oncocytoma RCCs and 1 of 1 (100%) case of collecting duct RCC. Strikingly, telomerase activity was significantly correlated with high grades (P = 0.000; Fig. 1B) and low stage clear cell RCC (P = 0.008; Fig. 1C). However, no significant difference in telomerase activity was observed among different subtypes of RCC (P = 0.355; Fig. 1D). Moreover, RCC samples from female showed significantly increased telomerase activity in comparison with RCC tissues from male (P = 0.034; Fig. 1E). On the other hand, no correlation of telomerase activity was found between different age groups of patients (P = 0.245). 3.3. Telomere length and its correlation with grade and biological age of patients Fig. 2 shows the mean telomere length in different RCC tissues. In most RCC tissues, significantly shorter telomere length were observed than that of adjacent normal renal parenchyma (P = 0.000; Fig. 3A). The median telomere length in the normal renal parenchyma and RCC tissues of all patients was found to be 10 kb (range 4.8 kb to 19.4 kb) and 5.4 kb (range 3.7 kb to 13.4 kb) respectively. We observed a significant correlation between telomere lengths in different grades of clear cell RCC. Low grade tumors (grade I & grade II: Median telomere length, 7.1 kb) had significantly longer telomeres than high grade tumors
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(grade III & grade IV: Median telomere length, 5.3 kb; P = 0.016; Fig. 3B). Notwithstanding, telomere length was not statistically correlated with the stage of RCC (P = 0.202; Fig. 3C), however telomere length was shorter in the low stage tumors (stage I & stage II: 5.75 kb) than high stage tumors (stage III & stage IV: 11.9 Kb). No significant correlation was found between telomere length and different subtypes of RCC (P = 0.669; Fig. 3D), (Table 1). Furthermore, no significant difference was observed between mean telomere length of male and female patients (P = 0.445; Fig. 3E). Additionally, telomere length was negatively correlated with the biological age of patients (P = 0.000; r2 = 0.528; Supplementary Fig. 1). 3.4. Prevalence of alternative lengthening mechanism in high stage tumors We extensively studied the prevalence of ALT mechanism in telomerase deficient RCC tissues by immunohistochemical staining of ALT associated PML bodies (Fig. 4). Evidence of ALT was noted in 8/10 (80%) of clear cell grade II, 2/3 (66.6%) of grade III, 2/2 (100%) of grade IV, 1/1 (100%) of sarcomatoid RCC and 2/3 (66.6%) of papillary RCC cases. Moreover, 12 of 28 (42.9%) cases of high stages of clear cell RCC, were telomerase negative. Among them, 11 were PML positive. On the other hand, only 3 of 41 (7.3%) cases of low stages of clear cell RCC were found to be telomerase negative. Out of these 3 cases of telomerase negative, only one case was found of PML positive. So, overall PML positivity was noticed in more cases of the high stages of RCC (11/28 (39.3%)) in comparison to low stages of RCC (1/41 (2.4%)) (P = 0.000). 4. Discussion RCC is the most lethal cancer of the urinary system. It is heterogeneous in reference to clinical behavior and prognosis. Telomere and telomerase biology play a critical and complex role in the initiation and progression of cancer. Henceforth, its better understanding may provide possible diagnostic and therapeutic implications. Strikingly, cancerous cells are distinguished from normal cells by genomic instability, uncontrolled proliferation and more rapid telomere shortening. It is notable that cancer cells become immortal when they reach a point of critical telomere shortening where telomerase might be activated to stabilize the telomeric repeats (Blackburn, 2010). The results of the present study demonstrated an advantage of measuring telomerase activity as it predicts the biological behavior of a tumor as supported by the present study, which succinctly showed that the telomerase activity was associated with histological grades and stages of RCC tumor, whereas telomerase activity was higher in high grade tumors (grade III & grade IV) as compared to low grade
Table 1 Relationship between relative telomerase activity, telomere length and clinicopathological features of renal cell carcinoma. Number
Relative telomerase activity, median value (range)
P-value
Mean telomere length, median value, (range) Kb
P-value
Gender Male Female
66 34
154 (43.6–641.5) 318 (116.3–8352)
0.034
6.2 (4.8–7.6) 4.9 (6.9–13.4)
0.445
Histological subtype Clear cell Papillary Sarcomatoid Chromophobe Oncocytoma Collecting duct
69 13 7 6 4 1
210 (0–8352) 74 (3–796) 148.5 (0–159) 164.1 (56–2843) 234.6 (192–278) 2
0.355
6.8 (2.7–13.4) 6.1 (4.8–7.4) 4.8 (4.7–4.9) 5.4 (4.2–6.4) 6.9 (5.8–7.5) 8.2
0.669
TNM stage (clear cell) Low stage (I & II) High stage (III & IV)
41 28
230.1 (93.5–8352) 30.3 (26.8–660.8)
0.008
5.75 (4.5–9.9) 11.9 (6–13.4)
0.202
Fuhrman grade (clear cell) Low grade (I & II) High grade (III & IV)
40 29
129 (35.7–487) 3423 (1245.8–8352)
0.000
7.1 (3.6–13.4) 5.3 (2.7–5.9)
0.016
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Fig. 1. Relative telomerase activity in (A) normal and tumor tissue and (B) different grades of clear cell RCC. Low grades consist of grades I & II and high grades consist of grades III & IV (C) different stages of clear cell RCC. Low stages consist of stages I & II and high stages consist of stages III & IV (D) Different types of RCC. 1, clear cell; 2, papillary; 3, sarcomatoid; 4, chromophobe; 5, oncocytoma; 6, collecting ducts (E). Genders of RCC patients. Telomerase activity was measured by TRAP assay. The extreme values were noted as dots and stars. The number next to the dots and stars represents the serial number of the sample. n, shows the number of cases in each group. Statistical analysis was done by Mann–Whitney and Kruskal–Wallis test. *P b 0.05 was considered as significant. **P b 0.01, ***P b 0.001.
Fig. 2. X-ray film showing mean telomere length. Chemiluminescent detection of mean telomere length with a DIG-labeled telomere-specific probe (TTAGGG)4. Lane MWM shows molecular weight marker. N, normal; T, tumor tissues. The data was analyzed as described in materials and methods resulting in the following mean TRF length of various samples: N1: 8.6 kbp; N2: 10.2 kbp; N3: 8.2 kbp; T1: 6.4 kbp; T2: 5.4 kbp; T3: 10.3 kbp; N4: 9.2 kbp; T4: 5.3 kbp; and T5: 5.6 kbp.
tumors (grade I & grade II). Conversely, it was higher in lower stage tumors (stage I & stage II) in comparison to high stage tumors (stage III & stage IV). This study first time portrayed the association between telomerase activity and tumor stages and grades, which was not reported in earlier studies related to RCC (Yoshida et al., 1998; Sugimura et al., 1999). However, several other studies have shown that telomerase activity was associated with histological tumor grades in other malignancies such as brain tumors (Hiraga et al., 1998), gastric cancer (Hu et al., 2009) and head and neck cancer (Zuzana et al., 2008). Telomerase activity has been defined as a marker of cancer aggressiveness and poor prognosis in non-small cell lung cancer, colorectal cancer, soft tissue sarcomas (Chen and Chen, 2011), and breast cancer (Poremba et al., 2000). However, we did not observe any significant change in telomerase activity among different subtypes of RCC which have different prognosis and aggressive behavior. Strikingly, telomerase activity was found to be significantly higher in female cases of RCC in comparison with male cases of RCC which is in accord to the previous study of Kyo et al. that could be associated with the activation of telomerase enzyme in response to estrogen levels (Kyo et al., 1999). The telomere length between male and female patients was not significantly different. Telomeres are critical for ensuring genome stability by preventing a loss of genetic information of cells. This maintenance role is usually accomplished by the enzyme telomerase, a reverse transcriptase complex that uses a short segment of its RNA subunit as a template to direct the addition of telomeric repeat onto chromosome ends (Xu et al., 2013). In the present study, mean telomere length was found to be significantly shorter in RCC tissues as compared to adjacent grossly normal renal tissues (Fig. 3A). Shortened telomeres have been associated with many cancers, including breast cancer (Poonepalli et al., 2008), pancreatic (Skinner et al., 2012), bladder (McGrath et al., 2007), lung (Frías et al., 2008) and T-cell prolymphocytic leukemia (Roth et al., 2007). However,
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Fig. 3. Mean telomere length in (A) normal and tumor tissue and (B) different grades of clear cell RCC. Low grades consist of grades I & II and high grades consist of grades III & IV (C) different stages of clear cell RCC. Low stages consist of stages I & II and high stages consist of stages III & IV (D). Different types of RCC. 1, clear cell; 2, papillary; 3, sarcomatoid; 4, chromophobe; 5, oncocytoma; 6, collecting ducts (E). Genders of RCC patients. Mean telomere length was measured by Southern blotting, hybridization using a telomeric probe (TTAGGG)4. The extreme values were noted as dots and stars. The number next to the dots and stars represents the serial number of sample. n, shows the number of cases in each group. Statistical analysis was done by Mann–Whitney and Kruskal–Wallis test. *P b 0.05 was considered as significant. ***P b 0.001; NS, Not significant.
telomere dysfunction resulting from replicative attrition constraint tumor growth by engaging DNA damage signaling pathways, sometimes promotes genomic instability and enhances carcinogenesis (Deng and Chang, 2007). Henceforth, critical telomere length is a decisive event which determines the fate of cell whether it should go for senescence or immortality by activating telomerase enzyme. In our series of samples, a trend displayed an association between telomere length and grading of RCC but it lacked with stages of tumor. Telomere shortening in high grade tumors is an indicator of extensive cell proliferation and increased telomerase activity represents as a compensatory mechanism for the critical maintenance of telomeres. Moreover, in this study, telomere length was found to be negatively correlated with biological age of the patients. Several previous studies also support our findings and showed that the average telomere length in cells from most human tissues decreases with age in vivo and with culture in vitro (Bojesen, 2013). Subsequently, telomere length could be a biomarker of biological age and thus indicate the risk of age related disease such as cancer, cardiovascular diseases, pulmonary disease and others (Bojesen, 2013). Thus, the present data underline the importance of telomere length as a diagnostic as well as biological marker. The previous studies also support our findings and showed that the average telomere length in cells from most human tissues decreases with age in vivo and in vitro (Henson et al., 2002). Surprisingly, we found that approximately 19% RCC cases lack detectable telomerase activity and a subset of these maintain telomere lengths by the telomerase independent telomere maintenance mechanism termed as an ALT. ALT positive carcinomas have shown to be arising from various tumors such as the bladder, cervix, endometrium, esophagus, gall bladder, kidney, liver and lung (Heaphy et al., 2011). Nevertheless, in some of these epithelial malignancies, they observed only a single ALT positive case; other carcinoma subtypes displayed considerable frequencies of ALT positivity. This study has shown for
the first time that the prevalence of ALT positive cases of RCC was significantly greater in high stages of RCC tumors in comparison with low stage tumor. It is not clear that activation of a telomere maintenance mechanism and immortalization are essential for all tumors. There are few cases where we could not find the telomerase activity as well as ALT positivity. In these cases of RCC, an unidentified mechanism may exist which needs to be explored. Usually, tumors with neither telomerase–telomere maintenance mechanism nor ALT-telomere maintenance mechanism are referred to as non-determined telomere maintenance mechanism (Hung et al., 2013). However, the presence of ALT positive was seen in all the grades of tumors as well as subtypes of RCC (Fig. 4). However, further studies are required to clarify the clinical and biological significance of ALT in the high stages of the RCC and its possible utility as a predictive and/or therapeutic target. Various studies have shown an association of ALT with different tumors and their clinicopathological features. A recent study by Mangerel et al., 2014 indicates that ALT positive tumors may be highly malignant and it was detected almost exclusively in malignant tumors. Similarly, another study (Costa et al., 2006) has also shown that ALT can support fully malignant liposarcomas and is associated with unfavorable disease outcome. Although ALT in tumors has not been studied extensively but in astrocytomas and osteosarcomas ALT is found to be common. Recently, a study by Liau et al., 2015 reported that ALT phenotype in the leiomyosarcoma is associated with aggressive histologic features and poor clinical outcome. The prognostic significance of ALT varies according to tumor type. The type of telomere maintenance mechanism used by tumors may have prognostic significance. Our findings support that telomere shortening and telomerase activation are responsible for the early development and aggressiveness of the tumor, but for the long term proliferation and metastasis of the RCC, the ALT mechanism may be responsible. In addition, telomerase activity and telomere length can be used as predictive and diagnostic markers. This study will also help in designing the targeted therapy against the
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Fig. 4. Immunohistochemical staining of ALT associated PML bodies in different grades and types of RCC. (A) Normal renal tissue showed no ALT associated PML body expression. (B) Grade II clear cell RCC, (C) grade III clear cell RCC, (D) grade IV clear cell RCC, (E) papillary RCC, (F) sarcomatoid RCC, (G) telomerase positive section of RCC-negative case of ALT associated PML bodies. No ALT associated PML bodies were detected. Arrows indicate ALT staining. Original magnification ×40.
telomerase negative tumors because anti-telomerase targeted therapy will not be effective against the tumors in which ALT mechanism prevails over telomerase activity. However, further study of a large number of patients in different subtypes of RCC is required to support a role of telomerase or the ALT pathway in different types of RCC. In addition, non-determined telomere maintenance mechanism needs to be explored in the cases where both telomerase activity as well as ALT mechanism is absent. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.02.079. Conflict of interest None. Acknowledgments This is a part of a research project funded by the Council of Scientific and Industrial Research, New Delhi, India (vide letter no. 27/0218/09/ EMR-II). The authors are thankful to Indian Council of Medical Research, New Delhi, India for awarding junior and senior research fellowship (3/ 1/3/JRF-2010/HRD-67 (11065)). References Allsopp, R.C., Chang, E., Kashefi-Aazam, M., Rogaev, E.I., Piatyszek, M.A., Shay, J.W., Harley, C.B., 1995. Telomere shortening is associated with cell division in vitro and in vivo. Exp. Cell Res. 220, 194–200. Artandi, S.E., DePinho, R.A., 2010. Telomeres and telomerase in cancer. Carcinogenesis 31, 9–18. Ausubel, M.F., Brent, R., Kingston, E.R., Seidman, J.G., Smith, A.J., Struhl, K., 2002. 5th ed. Short Protocols in Molecular Biology 1. John Wiley & Sons Inc., pp. 2–8. Blackburn, E.H., 1991. Structure and function of telomeres. Nature 350, 569–573.
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Augmented telomerase activity, reduced telomere length and the presence of alternative lengthening of telomere in renal cell carcinoma: Plausible predictive and diagnostic markers Deeksha Pal a, Ujjawal Sharma a, Ragini Khajuria a, Shrawan Kumar Singh b, Nandita Kakkar c, Rajendra Prasad a,⁎ a b c
Department of Biochemistry, PGIMER, Chandigarh, India Department of Urology, PGIMER, Chandigarh, India Department of Histopathology, PGIMER, Chandigarh, India
a r t i c l e
i n f o
Article history: Received 18 November 2014 Received in revised form 4 February 2015 Accepted 12 February 2015 Available online 11 March 2015 Keywords: Telomerase Telomere length PML bodies ALT TRAP
a b s t r a c t In this study, we analyzed 100 cases of renal cell carcinoma (RCC) for telomerase activity, telomere length and alternative lengthening of telomeres (ALT) using the TRAP assay, TeloTTAGGG assay kit and immunohistochemical analysis of ALT associated promyelocytic leukemia (PML) bodies respectively. A significantly higher (P = 0.000) telomerase activity was observed in 81 cases of RCC which was correlated with clinicopathological features of tumor for instance, stage (P = 0.008) and grades (P = 0.000) but not with the subtypes of RCC (P = 0.355). Notwithstanding, no correlation was found between telomerase activity and subtypes of RCC. Strikingly, the telomere length was found to be significantly shorter in RCC (P = 0.000) to that of corresponding normal renal tissues and it is well correlated with grades (P = 0.016) but not with stages (P = 0.202) and subtypes (P = 0.669) of RCC. In this study, telomere length was also negatively correlated with the age of patients (r2 = 0.528; P = 0.000) which supports the notion that it could be used as a marker for biological aging. ALT associated PML bodies containing PML protein was found in telomerase negative cases of RCC. It suggests the presence of an ALT pathway mechanism to maintain the telomere length in telomerase negative RCC tissues which was associated with high stages of RCC, suggesting a prevalent mechanism for telomere maintenance in high stages. In conclusion, the telomerase activity and telomere length can be used as a diagnostic as well as a predictive marker in RCC. The prevalence of ALT mechanism in high stages of RCC is warranted for the development of anti-ALT inhibitors along with telomerase inhibitor against RCC as a therapeutic approach. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Renal cell carcinoma (RCC) is the most prevalent and most lethal cancer of the kidney which accounts for approximately 3% of adult malignancies (Pal et al., 2014). A substantial gain in its incidence has been reported during the last decades (Siegel et al., 2014). Histopathologically, about 80% of RCCs are the clear cell type and the rest of the RCC subtypes include papillary, chromophobe & collecting duct. The clinical diagnosis of RCC is often confirmed by imaging studies, including Xray and computed tomography but the possible existence of benign renal tumor is a serious challenge to the diagnosis. Importantly, RCC is one of the most therapy resistant cancers. It responds very poorly or not at all to chemotherapy and hormonal and radiation therapies
Abbreviations: RCC, renal cell carcinoma; ALT, alternative lengthening of telomeres; PML, promyelocytic leukemia; TRAP, telomere repeat amplification protocol. ⁎ Corresponding author. E-mail address: [email protected] (R. Prasad).
http://dx.doi.org/10.1016/j.gene.2015.02.079 0378-1119/© 2015 Elsevier B.V. All rights reserved.
(Walsh et al., 2002). It is notable that a better understanding of RCC tumor biology at the molecular level is utmost important to improve current diagnosis, prognosis and treatment of RCC. One of the hallmarks of cancer is unlimited proliferation capacity, which is strictly associated with the ability to maintain telomeres through the activation of telomerase (Xu et al., 2013). Telomeres consist of 4 to 15 kb of the repetitive hexamer DNA sequence (TTAGGG) at the ends of chromosomes and undergo progressive shortening approximately 50–100 bp with each round of cell division (Blackburn, 1991). The repetitive noncoding telomeric repeats function as a buffer zone preventing the adjacent coding region of the genome from erosion by the telomeric DNA shortening. Telomerase, or telomere terminal transferase, is a ribonucleoprotein that involves in the de novo synthesis and elongation of telomeric repeats onto chromosomal ends by adding (TTAGGG)n repeats (Blackburn, 1991). Functionally, immortal germ cell lines express telomerase and maintain sufficient telomeric repeats, whereas most human somatic cells are unable to acquire telomerase activity in successive cell culture and become senescent (Allsopp et al., 1995).
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Stabilization of telomeres by telomerase reactivation appears to be concomitant with attainment of immortality which is likely to be necessary for infinite tumor growth. Most of the cancer cells maintain the telomere length with telomerase (Kim et al., 1994). Fewer yet significant numbers of tumor cells bypass the end replication in a telomerase independent manner, the process of which is called alternative lengthening of telomeres (ALT). Several lines of evidences indicate that an ALT involves a recombination based mechanism (Dunham et al., 2000) and characteristics of ALT cells include long and heterogeneous telomeres and subnuclear structure, termed as ALT associated promyelocyctic leukemia (PML) bodies (APBs). These structures contain telomeric DNA, telomere specific binding proteins (TRF1 & TRF2) and proteins involved in DNA replication and recombination (Cesare and Reddel, 2010). However, in few cases of cancers telomere maintenance mechanism is still not known (Ulaner et al., 2003). There are mounting evidences for the existence of a significant relationship between telomeres, telomerase and cancer and cancer mortality (Artandi and DePinho, 2010; Willeit et al., 2010; Shay and Wright, 2011). There are only few reports available in the literature on evaluation of telomerase activity (Yoshida et al., 1998; Fujioka et al., 2000) as well as telomere length in RCC (Mehle et al., 1994; Dahse et al., 1999). Approximately, 85% of all human tumors have telomerase activity (Hrdlicková et al., 2012) and specific cancer subset exhibits the ALT phenotype, a telomerase independent telomere maintenance mechanism (Nguyen et al., 2013). Nevertheless, the presence of ALT in human RCC has not been studied extensively. Moreover, assessments of telomerase activity, telomere length and ALT as well as their association with grades and stages have not been evaluated in RCC patients. In view of these facts, a comprehensive study was extended to interpret the status of telomerase activity, telomere length and ALT in RCC progression, diagnosis and the future treatment by targeting telomerase/telomere or ALT pathway. 2. Materials and methods
total protein extract for 20 min at 85 °C. The ELISA was performed in triplicate. The relative telomerase activities (RTA) of different samples were calculated byðAS−AS; OÞ=AS; IS x100 ðATS8−ATS8; 0Þ=ATS8; IS
RTA ¼ where
AS AS,0 AS,IS ATS8 ATS8,0 ATS8,IS
absorbance of the sample absorbance of heat treated samples absorbance of internal standard of the sample (IS) absorbance of control template (TS80) absorbance of lysis buffer absorbance of internal standard (IS) of the control template (TS8).
2.3. Telomere restriction fragment length analysis The genomic DNA was extracted using the standard method, with proteinase K treatment and phenol/chloroform extraction from 25 to 50 mg of the frozen tissue (Ausubel et al., 2002). Telomere length was assessed by measuring the telomere restriction fragments using the TeloTTAGGG telomere length assay kit (Roche Diagnostics, Indianapolis, USA) by following manufacturer's instructions. After digestion of the genomic DNA with Hinf1 and Rsa1 restriction enzymes, fragments were separated on a 0.8% agarose gel. The DNA was then transferred onto the nylon membrane (Roche Diagnostics, Indianpolis, USA) by Southern blotting and the blotted DNA fragments were hybridized with digoxigeninlabeled probe specific for telomeric repeats and incubated with a digoxigenin-specific antibody covalently coupled to alkaline phosphate. Finally, the immobilized telomere probe was visualized by metabolizing CDP-Star, a highly sensitive chemiluminescence substrate. The average TRF length was calculated by the following formula given in the kit.
2.1. Tissue specimens The present study was approved by the institute ethics committee and informed consent was obtained from patients. Following nephrectomy, tissue samples were taken from the tumor and grossly normal renal parenchyma separately. The samples were snap frozen in liquid nitrogen and stored at −80 °C till further use. Grading of tumors was done by the Fuhrman grading system (Fuhrman et al., 1982). 2.2. Telomerase activity by telomere repeat amplification protocol (TRAP) assay The tissue samples (50–100 mg) were homogenized and grinded after freezing in liquid nitrogen. The finely minced tissue was then extracted with lysis solution from the TeloTAGGG Telomerase PCR ELISAPLUS Kit (Roche Diagnostics, Indianapolis, USA) following the manufacturer's recommendations. The supernatant was transferred to a fresh tube and the total protein concentration was determined by the Lowry method (Lowry et al., 1951). Subsequently, TRAP reaction was carried out with 5 μg of total protein extract in a final volume of 50 μl, starting with the primer elongation step for 30 min at 25 °C, followed by telomerase inactivation for 5 min at 94 °C and 33 PCR cycles with denaturing for 30 s (94 °C), annealing for 30 s (50 °C), and polymerization for 90 s (72 °C). A 5 μl aliquot of the PCR product was denatured, hybridized to a DIG-labeled telomeric repeat-specific probe and immobilized onto a streptavidin-coated microtiter plate via the biotin-labeled synthetic PlTS primer used for PCR. The amount of immobilized PCR product was determined with a peroxidase-conjugated anti-DIG antibody followed by a 3,3′,5,5'-tetramethylbenzidine (TMB) substrate color reaction. Absorbance was measured at 450 nm (reference wavelength, 650 nm) using an ELISA reader. Negative controls were prepared by incubating the
TRF ¼
∑ðODi Þ ∑ðODi =Li Þ
where, ODi Li
chemiluminescent signal length of TRF at position i.
2.4. Immunohistochemical analysis of ALT associated PML bodies Goat polyclonal IgG antibody directed against the N-terminus of the human PML protein (Santa Cruz, USA) was used for immunostaining at 1:100 dilutions (Sharma et al., 2014). Briefly, 5 μm tissue sections were cut, deparaffinized in xylene followed by rehydration in 100%, 95% and 70% ethanol. Antigen retrieval was carried out by boiling the slides in 10 mM citrate buffer, pH 6.0 for 15 min. Slides were then treated with blocking serum for 10 min, after which they were incubated with primary antibody at 4 °C overnight in a humid chamber, followed by incubation with HRP anti-goat IgG (1:100) secondary antibody overnight in a humid chamber (Bangalore Geni, India). Chromogen detection was performed with diaminobenzidine (DAKO Corp., Carpinteria, CA) solution (0.5 ml of stock DAB in 4.5 ml of Tris buffer with 20 μl of hydrogen peroxide). Slides were counterstained with hematoxylin and photographed. 2.5. Statistical analysis Statistical evaluation was performed using SPSS program (version 20.0; SPSS Inc., Chicago, IL). Differences in mean telomerase activity as well as relative telomere length between two groups were evaluated using Mann–Whitney and between more than two groups were
D. Pal et al. / Gene 562 (2015) 145–151
evaluated using Kruskal–Wallis test. To analyze correlation between tumor grades, stages, subtypes of RCC and telomerase activity, telomere length Spearman rank correlation test was used. The correlation of PML positivity and high stages of RCC was evaluated by Pearson Chi-Square test. A P-value of b0.05 was considered significant. 3. Results 3.1. Patient's characteristics A total of 100 cases of histopathologically proven RCC were included in this study that includes 69 cases of clear cell carcinoma, 13 papillary, 7 sarcomatoid, 6 chromophobe, 4 oncocytoma and 1 of collecting duct carcinoma. Detailed patient's characteristics are given in Table 1. 3.2. Telomerase activity and its correlation with histological characteristics TRAP assay revealed detectable telomerase activity in 81% of the cases of RCC and it was found to be significantly higher to that of adjacent normal renal parenchyma (P = 0.000; Fig. 1A). Telomerase activity was present in 54 of 69 clear cell RCCs (78.3%), 10 of 13 cases of papillary RCCs (76.9%), 6 of 7 (85.7%) cases of sarcomatoid RCCs, 6 of 6 (100%) cases of chromophobe RCCs, 4 of 4 (100%) cases of oncocytoma RCCs and 1 of 1 (100%) case of collecting duct RCC. Strikingly, telomerase activity was significantly correlated with high grades (P = 0.000; Fig. 1B) and low stage clear cell RCC (P = 0.008; Fig. 1C). However, no significant difference in telomerase activity was observed among different subtypes of RCC (P = 0.355; Fig. 1D). Moreover, RCC samples from female showed significantly increased telomerase activity in comparison with RCC tissues from male (P = 0.034; Fig. 1E). On the other hand, no correlation of telomerase activity was found between different age groups of patients (P = 0.245). 3.3. Telomere length and its correlation with grade and biological age of patients Fig. 2 shows the mean telomere length in different RCC tissues. In most RCC tissues, significantly shorter telomere length were observed than that of adjacent normal renal parenchyma (P = 0.000; Fig. 3A). The median telomere length in the normal renal parenchyma and RCC tissues of all patients was found to be 10 kb (range 4.8 kb to 19.4 kb) and 5.4 kb (range 3.7 kb to 13.4 kb) respectively. We observed a significant correlation between telomere lengths in different grades of clear cell RCC. Low grade tumors (grade I & grade II: Median telomere length, 7.1 kb) had significantly longer telomeres than high grade tumors
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(grade III & grade IV: Median telomere length, 5.3 kb; P = 0.016; Fig. 3B). Notwithstanding, telomere length was not statistically correlated with the stage of RCC (P = 0.202; Fig. 3C), however telomere length was shorter in the low stage tumors (stage I & stage II: 5.75 kb) than high stage tumors (stage III & stage IV: 11.9 Kb). No significant correlation was found between telomere length and different subtypes of RCC (P = 0.669; Fig. 3D), (Table 1). Furthermore, no significant difference was observed between mean telomere length of male and female patients (P = 0.445; Fig. 3E). Additionally, telomere length was negatively correlated with the biological age of patients (P = 0.000; r2 = 0.528; Supplementary Fig. 1). 3.4. Prevalence of alternative lengthening mechanism in high stage tumors We extensively studied the prevalence of ALT mechanism in telomerase deficient RCC tissues by immunohistochemical staining of ALT associated PML bodies (Fig. 4). Evidence of ALT was noted in 8/10 (80%) of clear cell grade II, 2/3 (66.6%) of grade III, 2/2 (100%) of grade IV, 1/1 (100%) of sarcomatoid RCC and 2/3 (66.6%) of papillary RCC cases. Moreover, 12 of 28 (42.9%) cases of high stages of clear cell RCC, were telomerase negative. Among them, 11 were PML positive. On the other hand, only 3 of 41 (7.3%) cases of low stages of clear cell RCC were found to be telomerase negative. Out of these 3 cases of telomerase negative, only one case was found of PML positive. So, overall PML positivity was noticed in more cases of the high stages of RCC (11/28 (39.3%)) in comparison to low stages of RCC (1/41 (2.4%)) (P = 0.000). 4. Discussion RCC is the most lethal cancer of the urinary system. It is heterogeneous in reference to clinical behavior and prognosis. Telomere and telomerase biology play a critical and complex role in the initiation and progression of cancer. Henceforth, its better understanding may provide possible diagnostic and therapeutic implications. Strikingly, cancerous cells are distinguished from normal cells by genomic instability, uncontrolled proliferation and more rapid telomere shortening. It is notable that cancer cells become immortal when they reach a point of critical telomere shortening where telomerase might be activated to stabilize the telomeric repeats (Blackburn, 2010). The results of the present study demonstrated an advantage of measuring telomerase activity as it predicts the biological behavior of a tumor as supported by the present study, which succinctly showed that the telomerase activity was associated with histological grades and stages of RCC tumor, whereas telomerase activity was higher in high grade tumors (grade III & grade IV) as compared to low grade
Table 1 Relationship between relative telomerase activity, telomere length and clinicopathological features of renal cell carcinoma. Number
Relative telomerase activity, median value (range)
P-value
Mean telomere length, median value, (range) Kb
P-value
Gender Male Female
66 34
154 (43.6–641.5) 318 (116.3–8352)
0.034
6.2 (4.8–7.6) 4.9 (6.9–13.4)
0.445
Histological subtype Clear cell Papillary Sarcomatoid Chromophobe Oncocytoma Collecting duct
69 13 7 6 4 1
210 (0–8352) 74 (3–796) 148.5 (0–159) 164.1 (56–2843) 234.6 (192–278) 2
0.355
6.8 (2.7–13.4) 6.1 (4.8–7.4) 4.8 (4.7–4.9) 5.4 (4.2–6.4) 6.9 (5.8–7.5) 8.2
0.669
TNM stage (clear cell) Low stage (I & II) High stage (III & IV)
41 28
230.1 (93.5–8352) 30.3 (26.8–660.8)
0.008
5.75 (4.5–9.9) 11.9 (6–13.4)
0.202
Fuhrman grade (clear cell) Low grade (I & II) High grade (III & IV)
40 29
129 (35.7–487) 3423 (1245.8–8352)
0.000
7.1 (3.6–13.4) 5.3 (2.7–5.9)
0.016
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Fig. 1. Relative telomerase activity in (A) normal and tumor tissue and (B) different grades of clear cell RCC. Low grades consist of grades I & II and high grades consist of grades III & IV (C) different stages of clear cell RCC. Low stages consist of stages I & II and high stages consist of stages III & IV (D) Different types of RCC. 1, clear cell; 2, papillary; 3, sarcomatoid; 4, chromophobe; 5, oncocytoma; 6, collecting ducts (E). Genders of RCC patients. Telomerase activity was measured by TRAP assay. The extreme values were noted as dots and stars. The number next to the dots and stars represents the serial number of the sample. n, shows the number of cases in each group. Statistical analysis was done by Mann–Whitney and Kruskal–Wallis test. *P b 0.05 was considered as significant. **P b 0.01, ***P b 0.001.
Fig. 2. X-ray film showing mean telomere length. Chemiluminescent detection of mean telomere length with a DIG-labeled telomere-specific probe (TTAGGG)4. Lane MWM shows molecular weight marker. N, normal; T, tumor tissues. The data was analyzed as described in materials and methods resulting in the following mean TRF length of various samples: N1: 8.6 kbp; N2: 10.2 kbp; N3: 8.2 kbp; T1: 6.4 kbp; T2: 5.4 kbp; T3: 10.3 kbp; N4: 9.2 kbp; T4: 5.3 kbp; and T5: 5.6 kbp.
tumors (grade I & grade II). Conversely, it was higher in lower stage tumors (stage I & stage II) in comparison to high stage tumors (stage III & stage IV). This study first time portrayed the association between telomerase activity and tumor stages and grades, which was not reported in earlier studies related to RCC (Yoshida et al., 1998; Sugimura et al., 1999). However, several other studies have shown that telomerase activity was associated with histological tumor grades in other malignancies such as brain tumors (Hiraga et al., 1998), gastric cancer (Hu et al., 2009) and head and neck cancer (Zuzana et al., 2008). Telomerase activity has been defined as a marker of cancer aggressiveness and poor prognosis in non-small cell lung cancer, colorectal cancer, soft tissue sarcomas (Chen and Chen, 2011), and breast cancer (Poremba et al., 2000). However, we did not observe any significant change in telomerase activity among different subtypes of RCC which have different prognosis and aggressive behavior. Strikingly, telomerase activity was found to be significantly higher in female cases of RCC in comparison with male cases of RCC which is in accord to the previous study of Kyo et al. that could be associated with the activation of telomerase enzyme in response to estrogen levels (Kyo et al., 1999). The telomere length between male and female patients was not significantly different. Telomeres are critical for ensuring genome stability by preventing a loss of genetic information of cells. This maintenance role is usually accomplished by the enzyme telomerase, a reverse transcriptase complex that uses a short segment of its RNA subunit as a template to direct the addition of telomeric repeat onto chromosome ends (Xu et al., 2013). In the present study, mean telomere length was found to be significantly shorter in RCC tissues as compared to adjacent grossly normal renal tissues (Fig. 3A). Shortened telomeres have been associated with many cancers, including breast cancer (Poonepalli et al., 2008), pancreatic (Skinner et al., 2012), bladder (McGrath et al., 2007), lung (Frías et al., 2008) and T-cell prolymphocytic leukemia (Roth et al., 2007). However,
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Fig. 3. Mean telomere length in (A) normal and tumor tissue and (B) different grades of clear cell RCC. Low grades consist of grades I & II and high grades consist of grades III & IV (C) different stages of clear cell RCC. Low stages consist of stages I & II and high stages consist of stages III & IV (D). Different types of RCC. 1, clear cell; 2, papillary; 3, sarcomatoid; 4, chromophobe; 5, oncocytoma; 6, collecting ducts (E). Genders of RCC patients. Mean telomere length was measured by Southern blotting, hybridization using a telomeric probe (TTAGGG)4. The extreme values were noted as dots and stars. The number next to the dots and stars represents the serial number of sample. n, shows the number of cases in each group. Statistical analysis was done by Mann–Whitney and Kruskal–Wallis test. *P b 0.05 was considered as significant. ***P b 0.001; NS, Not significant.
telomere dysfunction resulting from replicative attrition constraint tumor growth by engaging DNA damage signaling pathways, sometimes promotes genomic instability and enhances carcinogenesis (Deng and Chang, 2007). Henceforth, critical telomere length is a decisive event which determines the fate of cell whether it should go for senescence or immortality by activating telomerase enzyme. In our series of samples, a trend displayed an association between telomere length and grading of RCC but it lacked with stages of tumor. Telomere shortening in high grade tumors is an indicator of extensive cell proliferation and increased telomerase activity represents as a compensatory mechanism for the critical maintenance of telomeres. Moreover, in this study, telomere length was found to be negatively correlated with biological age of the patients. Several previous studies also support our findings and showed that the average telomere length in cells from most human tissues decreases with age in vivo and with culture in vitro (Bojesen, 2013). Subsequently, telomere length could be a biomarker of biological age and thus indicate the risk of age related disease such as cancer, cardiovascular diseases, pulmonary disease and others (Bojesen, 2013). Thus, the present data underline the importance of telomere length as a diagnostic as well as biological marker. The previous studies also support our findings and showed that the average telomere length in cells from most human tissues decreases with age in vivo and in vitro (Henson et al., 2002). Surprisingly, we found that approximately 19% RCC cases lack detectable telomerase activity and a subset of these maintain telomere lengths by the telomerase independent telomere maintenance mechanism termed as an ALT. ALT positive carcinomas have shown to be arising from various tumors such as the bladder, cervix, endometrium, esophagus, gall bladder, kidney, liver and lung (Heaphy et al., 2011). Nevertheless, in some of these epithelial malignancies, they observed only a single ALT positive case; other carcinoma subtypes displayed considerable frequencies of ALT positivity. This study has shown for
the first time that the prevalence of ALT positive cases of RCC was significantly greater in high stages of RCC tumors in comparison with low stage tumor. It is not clear that activation of a telomere maintenance mechanism and immortalization are essential for all tumors. There are few cases where we could not find the telomerase activity as well as ALT positivity. In these cases of RCC, an unidentified mechanism may exist which needs to be explored. Usually, tumors with neither telomerase–telomere maintenance mechanism nor ALT-telomere maintenance mechanism are referred to as non-determined telomere maintenance mechanism (Hung et al., 2013). However, the presence of ALT positive was seen in all the grades of tumors as well as subtypes of RCC (Fig. 4). However, further studies are required to clarify the clinical and biological significance of ALT in the high stages of the RCC and its possible utility as a predictive and/or therapeutic target. Various studies have shown an association of ALT with different tumors and their clinicopathological features. A recent study by Mangerel et al., 2014 indicates that ALT positive tumors may be highly malignant and it was detected almost exclusively in malignant tumors. Similarly, another study (Costa et al., 2006) has also shown that ALT can support fully malignant liposarcomas and is associated with unfavorable disease outcome. Although ALT in tumors has not been studied extensively but in astrocytomas and osteosarcomas ALT is found to be common. Recently, a study by Liau et al., 2015 reported that ALT phenotype in the leiomyosarcoma is associated with aggressive histologic features and poor clinical outcome. The prognostic significance of ALT varies according to tumor type. The type of telomere maintenance mechanism used by tumors may have prognostic significance. Our findings support that telomere shortening and telomerase activation are responsible for the early development and aggressiveness of the tumor, but for the long term proliferation and metastasis of the RCC, the ALT mechanism may be responsible. In addition, telomerase activity and telomere length can be used as predictive and diagnostic markers. This study will also help in designing the targeted therapy against the
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Fig. 4. Immunohistochemical staining of ALT associated PML bodies in different grades and types of RCC. (A) Normal renal tissue showed no ALT associated PML body expression. (B) Grade II clear cell RCC, (C) grade III clear cell RCC, (D) grade IV clear cell RCC, (E) papillary RCC, (F) sarcomatoid RCC, (G) telomerase positive section of RCC-negative case of ALT associated PML bodies. No ALT associated PML bodies were detected. Arrows indicate ALT staining. Original magnification ×40.
telomerase negative tumors because anti-telomerase targeted therapy will not be effective against the tumors in which ALT mechanism prevails over telomerase activity. However, further study of a large number of patients in different subtypes of RCC is required to support a role of telomerase or the ALT pathway in different types of RCC. In addition, non-determined telomere maintenance mechanism needs to be explored in the cases where both telomerase activity as well as ALT mechanism is absent. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.02.079. Conflict of interest None. Acknowledgments This is a part of a research project funded by the Council of Scientific and Industrial Research, New Delhi, India (vide letter no. 27/0218/09/ EMR-II). The authors are thankful to Indian Council of Medical Research, New Delhi, India for awarding junior and senior research fellowship (3/ 1/3/JRF-2010/HRD-67 (11065)). References Allsopp, R.C., Chang, E., Kashefi-Aazam, M., Rogaev, E.I., Piatyszek, M.A., Shay, J.W., Harley, C.B., 1995. Telomere shortening is associated with cell division in vitro and in vivo. Exp. Cell Res. 220, 194–200. Artandi, S.E., DePinho, R.A., 2010. Telomeres and telomerase in cancer. Carcinogenesis 31, 9–18. Ausubel, M.F., Brent, R., Kingston, E.R., Seidman, J.G., Smith, A.J., Struhl, K., 2002. 5th ed. Short Protocols in Molecular Biology 1. John Wiley & Sons Inc., pp. 2–8. Blackburn, E.H., 1991. Structure and function of telomeres. Nature 350, 569–573.
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