Cancer Letters, 62 (1992)
95 - 105
95
Elsevier Scientific Publishers Ireland Ltd.
Sensitivity of nuclear c-myc levels and induction to differentiation-inducing agents in human colon tumor cell lines C.W. Taylora, Y.S. Kimb, K.E. Childress-Fields” of Pharmacology.
‘Department Veterans
Administration
(Received
21 June
(Accepted
17 October
Baylor
Medical
College
Center.
of Medicine.
4150
Clement
and L.C. Yeomana
Houston.
Street,
San
TX
Six human colon tumor cell lines were analyzed for their constitutiue leuels of the c-myc protein. The nuclear proto-oncogene, c-myc, was detected as an expressed product in all of the human colon tumor cell lines analyzed.
levels
poorly differentiated cell lines HCTl16, and C showed c-myc leuels that aueraggreater
counterparts, c-myc levels were
oic acid,
colon
their
sodium
butyrate
and
TGF-/3,
of sensitiuity and resistance c-myc leuels were reduced cell phenotypes
formamide
treated
or sodium
with
dimethyl-
Only the well-
butyrate.
Only one of the human
lines
(GEO)
responded
Correspondence Abbreuiotions: mamide;
DOC,
colon
to
tumor
retinoic
cell
acid.
In-
to’ Lynn C. Yeoman. CPM.
counts per minute;
deoxycholate;
EGF.
FBS. fetal bovine serum; HCT, phenylmethanesulfonyl radioimmunoassay; transforming
growth
SDS,
sodium
factor-beta
0
1992
Printed and Published in Ireland
DMF.
epidermal
human
fluoride;
ethylaminomethane.
0304.3835/92/$05.00
Lab..
levels of c-myc protein were found to correlate well with greater growth rates and with poor differentiation class. Similarly, a parallel sensitivity to down-regulation of c-myc and
attenuation
curues for inducers observed in growth
of
c-myc
induction
of differentiation sensitive human
were colon
Keywords: c-myc; colon; oncogene; oncogene, induction and differentiation
protoagents
tumor
cell lines.
RA.
colon tumor:
dodecyl and
dimethylforgrowth factor:
retinoic
PMSF.
acid:
sulfate:
TRIS.
Introduction
distinct
emerged. in all the
differentiated human colon tumor cell lines were responsiue to transforming growth factorfl.
Research
(U.S.A.)
well-differentiated
in the presence of inducers of i.e., dimethylformamide, retin-
analyzed
patterns Nuclear
than
i.e., GEO, CBS and FET. When and responses to serum induction
differentiation,
‘Gastrointestinal 94121
1991)
creased
ed Z-fold
and CA
1991)
Summary
The MO
77030
Francisco,
RIA.
TGF-8.
trihydroxy-
In previous studies we have used immunological probes to analyze the cellular antigens expressed by a group of highly characterized human colon tumor cell lines [29]. These human colon tumor cell lines have been divided into three groups based upon their biological characteristics. Group 1 cell lines produce poorly differentiated tumors in athymic nude mice, grow we11 in soft agarose and secrete low levels of high molecular weight mucins into their media. Group III cell lines produce well differentiated zenografts in athymic nude mice, do not grow as efficiently in soft agarose, but secrete high levels of carcinoembryonic antigen into their culture medium. Group II cell lines fall between Groups I and III in their biological properties. The ex-
Elsevier Scientific Publishers Ireland Ltd
96
pression of specific tumor associated antigens revealed patterns of cytosolic antigen expression that correlated with the differentiation class of the individual cell lines [29]. In studies designed to examine the epiderma1 growth factor sensitivity of colon tumor cells we have shown that their growth factor to exogenous EGF’ and their responses numbers of cell surface EGF-receptors correlate with cell grouping [31]. More differentiated cell lines express more cell membrane associated EGF-receptor and show a substantial increase in growth response to exogenous EGF, whereas poorly differentiated colon tumor cell lines are insensitive to exogenous growth factor and exhibit low levels of cell membrane associated EGF-receptor. There is a good correlation between tumor associated antigen expression, growth factor receptor expression and cell phenotype in our cell line model system for human colon cancer [4,5]. Because of the reported induction of c-myc transcripts in response to growth signals [18] we have evaluated the levels of c-myc expression in this series of well characterized human colon tumor cell lines. The c-myc protein is a 439-amino acid phosphoprotein of 51 kDa molecular weight that has been shown to migrate on SDS containing polyacrylamide gels as a 62/64 kDa band [1,2,8,15]. Using various inducers of differentiation in the study of malignant hematopoietic cells the c-myc nuclear proto-oncogene has been shown to immortalize and/or transform cells as a primary oncogene [26]. It mediates signal transduction in the cell nucleus through its DNA-binding activity [8]. The net effect of c-myc on transformation and tumor progression is thought to occur as a result of proliferative responses to c-myc expression. This is thought to cause an induction of replication competence during the cell cycle [17]. Studies on the effects of inducers of differentiation in malignant hematopoietic cells suggest that c-myc expression is decreased during the induction of differentiation [9,25,32]. The importance of understanding c-myc expression at the protein level is illustrated by the
complex manner in which c-myc levels are regulated. The level of c-myc expression has been shown to be regulated at the point of transcription initiation, transcriptional elongation, mRNA stability and protein translation
171. There appears to be a strong correlation between tumorigenicity and cell grouping in the human colon tumor lines we have studied. Furthermore, reports from other laboratories have described correlations between c-myc level, malignancy and tumor size in clinical studies on human colorectal and gastric cancer [6,11,16,27,28]. We became interested, therefore, in determining whether c-myc level is related to cell grouping in this set of characterized human colon tumor cell lines. Finley et al. [14] found modest c-myc overexpression in approximately two-thirds of the colorectal carcinoma cases that they examined. These reports are consistent with those made earlier by Erisman et al. [lo] who showed that expression of the c-myc gene at both the mRNA and protein level occurs constitutively and is 8- to 37-fold higher than levels of c-myc protein in normal mucosal cells. The predictive use of c-myc levels in clinical specimens, however, has not proved useful as a correlate for clinical outcome in malignancies of the colon [lo]. We have hypothesized that analyses performed in a set of defined colon cell lines that exhibit a wide range of biological characteristics could be very useful. It has been shown that c-myc transcripts can be downregulated by a variety of different agents in a large number of cell types [24,33]. By using differentiation-inducing chemicals and peptides, the influence of these agents on c-myc levels in colon cell lines of defined differentiation phenotype could be determined. The use of colon tumor cell lines avoids the clonal heterogeneity associated with the study of solid tumors. Similarly, the influence of invading cells can be eliminated. Because of the complex nature of the regulation of c-myc expression, determining the protein level of the c-myc proto-oncogene product is probably the only reliable indicator of c-
myc function in growth and differentiation [26]. We have analyzed the constitutive level of c-myc expression in six colon cell lines. Three were selected from each of the two most divergent phenotype classes [4,5]. By examining constitutive c-myc levels, determining cmyc enhancement following serum induction and analyzing the effects of differentiationinducing agents on c-myc levels and cell growth rates we have established a clear relationship between c-myc level and cell phenotype in human colon tumor cells. Materials and Methods Tissue culture and cell fines The establishment and characterization of a series of human colon tumor cell lines that were derived from primary colon tumors have been described previously [3]. These cell lines have been divided into three groups based upon their in vitro biological characteristics and tumorigenicity in athymic nude mice [4,5]. These studies were performed on cells grown: (i) in supplemented McCoy’s 5A medium containing 10 percent FBS, (ii) starved for 4 days in the presence of supplemented McCoy’s 5A medium and (iii) re-fed with 10% FBS in supplemented McCoy’s 5A medium. Mitogenesis assay Cells were seeded at densities of 20 000 cells per 35-mm well in McCoy’s 5A medium containing 10% FBS. Cells were grown to 75% confluence and starved for 5 days. Cells were given a l-h pulse with [“Hlthymidine 50 Ci/mmole; ICN (100 ~1; 25 &i; Radiochemicals, Irvine, CA) at 37OC [22] after 0, 1, 2, 3, 4 and 5 days of starvation. Thymidine-containing medium was removed and the cells were washed with 1.0 ml of plain McCoy’s 5A medium. Cell membranes were lysed by incubation for 10 min with 10% trichloroacetic acid at 4OC and washed once with trichloroacetic acid. The contents of each well were removed with 0.5 ml of 0.2 M NaOH containing 40 pg/ml calf thymus DNA. Samples were analyzed for [3H]thymidine-
labeled DNA by counting in a Beckman ~~7500 liquid scintillation counter (Beckman Instruments; Palo Alto, CA). Serum starvation and induction of c-myc Cell lines were plated and grown from 70-80% confluence in supplemented McCoy’s 5A medium containing 10% fetal bovine serum (10F). Cells were subsequently starved by incubation for 96 h in McCoy’s 5A medium. Cells were fed again at time zero (T = 0) with media containing 10% fetal bovine serum in the presence or absence of TGF-P (10 ng/ml), retinoic acid (1 .O PM), dimethylformamide (1.0%) or sodium butyrate (2 mM). Preparation of whole cell Iysates and crude nuclei Cells were detached by scraping and washing with phosphate buffered saline containing 1 .O mM phenylmethanesulfonyl fluoride (PMSF). Washed cells were suspended in a modified Laemmli sample buffer [19] containing 1% SDS, 10% glycerol, 0.2 M Tris-HCI (pH 6.8), 1.0 mM PMSF, 0.1 mM leupeptin and 0.1 mM aprotinin. Cells were repeatedly dispersed and homogenized by sequential passage through 18-, 21. and 25-gauge syringe needles. Homogenization was repeated until the solution was no longer viscous. Crude nuclei were prepared by the low salt procedure of Evans and Hancock [13]. Cells were washed with PBS and suspended in low salt lysis buffer containing 20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 mM KCI, 5 mM MgC&, 0.5% Nonidet P-40, 0.1% DOC, 1.0 mM PMSF, 0.1 mM leupeptin and 0.1 mM aprotinin. After mixing by repeated pipetting, cells were lysed for 5 min at 4OC with intermittent mixing. Nuclei were collected by centrifugation at 1000 X g for 5 min and washed in lysis buffer before suspension in a modified Laemmli sample buffer [19]. Polyacrylamide gel electrophoresis and immunoblotting Poiyacrylamide gel electrophoresis was per-
98
formed as described by Laemmli [ 191. Precisely 75 pg of protein lysate, containing bromophenol blue and 2-mercaptoethanol, were loaded into each sample well of a 10% polyacrylamide SDS slab gel. Electrophoresis was performed in a Bio-Rad Protean Cell (BioRad; Richmond, CA) and continued at 150 V until the bromophenol blue dye front was within 0.5 cm of the end of the gel (approximately 2.5 -3.0 h). Proteins were electrophoretically transferred to nitrocellulose paper in a Bio-Rad Trans-Blot Cell at a current of 250 mA for 4 h as described by Towbin et al. [30]. Prior to immunoblotting the nitrocellulose sheets were blocked with a PBS solution containing 3% RIA grade BSA (Sigma, St. Louis, MO) and 0.5% Tween-20. Immunodetection was achieved using a pan reactive anti-c-myc antibody (sheep IgG) obtained from Cambridge Research Biochemicals (Valley Stream, NY). The primary antibody was used at a 1:lOO dilution for 2 h at 25OC; the rabbit anti-sheep second antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1:500 dilution for 2 h at 25OC and ‘251-protein G (DuPont Company; Boston, MA) at a concentration of 1 x lo5 cpm/ml for 2 h at 25OC. All antibody and protein G solutions were diluted in a PBS solution containing 1% RIA grade BSA and 0.5% Tween-20. All blocking and immunoblot steps were performed with gentle mixing (75 rev./min) on a Gyratory Shaker (New BrunsEdison, NJ) wick Scientific Instruments, Autoradiographic patterns were obtained by imexposing developed, nitrocellulose munoblots to Kodak X-Omat AR-5 film with Du Pont Chronex intensifier screens (Du Pont Company; Boston, MA) for 1 - 4 h. Quantitative
comparison
of c-myc
levels
In preparation for quantitation the corners of immunoblotted nitrocellulose sheets were spotted with r4C-labeled radioactive ink to permit the precise alignment of Xerox transparency sheets of autoradiograms with the nitrocellulose blots. Following alignment of the xerographic pattern with the nitrocellulose
sheet, the 62 kDa band was outlined, cut out and counted in a Beckman 4000 gamma counter (Beckman Instruments, Palo Alto, CA). Samples were counted for 10 min or 100 000 counts, whichever came first and corrected for background. Background values were obtained for several pieces of nitrocellulose of comparable area cut from regions of the nitrocellulose sheet that were devoid of visually detectable autoradiographic density and averaged. Results Specificity
of the c-myc
molecular
weight
tein
and
human
constitutiue colon
polyclonal
behavior
tumor
of the
c-myc
protein
antibody, c-myc
pro-
levels
in
cell lines
To establish that the immunoreactive bands detected in whole cell extracts and crude nuclear fractions prepared from human colon tumor cell lines were authentic c-myc gene products, immunoblots were performed using both c-myc reactive and antibody that had been preabsorbed with a lo-fold excess of immunizing peptide. The immunoblot pattern obtained when nuclear fractions from HCTl16, RKO, C, FET, GE0 and GLY cells were immunoblotted is shown in Fig. 1. Preabsorption with immunizing peptide eliminated the detection of the authentic 62-kDa c-myc band (data not shown). A molecular weight of 62 kDa was calculated for the single immunoreactive band. This value was based upon the migration of the immunoreactive band relative to protein standards of known molecular weight and agrees well with the reported molecular weight behavior of c-myc [1,2,8,15] Measuring the level of ‘251-protein G binding to the respective lanes shown in Fig. 1 revealed that c-myc protein levels were uniformly higher in poorly differentiated colon tumor cell cultures (Group III cell lines: HCT116, RKO and C) when compared to well-differentiated cell lines (Group I cell lines: FET, GE0 and GLY). The average c-myc level (14 400 cpm) in a poorly differentiated colon tumor cell line was 1.7 times higher than
99
_._
7 0
Y
62k
-45
E 0” cl .g
1.5~-
1.0-m
b k
-31
&
0.5-m
3 x2 0.01
RKO
HCT116
C
GE0
GLY
FEX
HCT Cell Line
HCT.116 RKO -II
Group I
C
FET
GE0
GLY
Group III
Fig. 1. Quantitative immunoblot analysis of human colon tumor cell nuclear extracts with anti-c-myc polyclonal antibody. Nuclear fractions were prepared from Group I (HCT116, RKO and C) cells and Group III (FET, GE0 and GLY) cells and 75 pg loads subjected to electrophoresis on 10% Laemmli gels [19]. Protein was transferred to nitrocellulose sheets according to the method of Towbin [30] and immunoreactivity probed with polyclonal anti-c-myc antibody. Immunoreactive bands were detected using 1251-labeled protein G and autoradiography. The c-myc protein band is marked with an arrow at 62 kDa.
the corresponding average level determined for well differentiated colon cells (8500 cpm). The data shown in the bar graph in Fig. 2 indicate that the range of ‘251-protein G counts associated with the c-myc band extended from 7500 cpm for FET cells to 18 000 cpm for RKO cells. Influence tumor
of sodium butyrate on human colon
cell growth
Before the influence of sodium butyrate concentration on c-myc levels could be addressed, it was necessary to determine the influence of butyrate dose on colon tumor cell growth. Both HCT116 cells and FET cells were sub-
Fig. 2. Comparison of natural c-myc levels in six human colon tumor cell lines. Crude nuclei were prepared from RKO, HCT116, C, GEO. GLY and FET cells and subjected to polyacrylamide gel electrophoresis and immunoblotting. Binding of ‘251-labeled protein G to sheep polyclonal anti-c-myc antibody on nitrocellulose sheets was determined by cutting autoradiograph localized bands from the nitrocellulose sheet and counting in a gamma counter.
jetted to growth for 5 days in the presence of sodium butyrate concentrations ranging from 0 - 4 mM. Additions of sodium butyrate were made on days 1 and 3 when culture media was changed. The different percentages of inhibition of colon tumor cell line growth by sodium butyrate are shown in Fig. 3. Both HCT116 (poorly-differentiated) and FET (well-differentiated) cells were inhibited by sodium butyrate. IC50 values of 0.34 mM and 0.80 mM were calculated for HCT116 and FET respectively. The appearance of cells, HCT116 and FET cells after being grown in the presence of 2 mM sodium butyrate for 5 days is shown in Fig. 4. Because there was a substantial reduction in the number of cells counted in cultures grown in the presence of sodium butyrate, the question of butyrate toxicity was addressed. Staining of 2 mM sodium butyrate-treated and -untreated control cultures with Trypan Blue did not reveal a significant difference in cell viability (I 2 - 5% dye uptake) between butyrate-treated and control cell cultures (data not shown).
c-myc levels reduced from 42 - 78% of control for TGF-fl and 12 -30% of control for retinoic acid (when the influence of RA on FET cells is excluded). Cellular responses to DMF were more prevalent, i.e., DMF caused decreases ranging from 5 - 27% in poorlydifferentiated cell lines and 29 - 75% in welldifferentiated cells. Sodium butyrate, on the other hand, showed a strong effect upon cmyc levels in both poor- and well-differentiated human colon tumor cell lines. Poorly-differentiated cell lines showed 36 - 72% reductions in c-myc level while well-differentiated lines ranged from 49-59%. Induction
0
1 Sodium
2 Butyrate
3
4
(mM)
Fig. 3. Inhibition of HCT116 and FET cell growth by treatment with different concentrations of sodium butyrate. Human colon tumor cell lines were seeded at a density of 1 x lo6 cells per well in 6-well plates and permitted to attach for 24 h. Media were changed and replaced with fresh media containing 0.5, 1.0, 1.5, 2.0 and 4.0 mM sodium butyrate. Fresh media containing equivalent concentrations of sodium butyrate were added again on day 3 and cells counted on day 5. Results are presented as percentage inhibition with 100% based upon a 0.0 mM sodium butyrate control.
It has already been shown that DMF inhibits growth in a variety of human colon tumor cell lines [ZO], TGF-0 effects are limited to welldifferentiated cell line members of Group 111 and retinoic acid has little or no influence on colon tumor cell line growth [21].
refeeding in cell lines To determine the c-myc responses that accompany serum re-feeding, HCTl16 cells from Group I and FET cells from Group III were seeded and grown to 75% confluence in the presence of 10% FBS before starving for 4 days. Measurement of the fall in [3H]thymidine incorporation into DNA was used to establish the appropriate length of the starvation period. The c-myc levels were measured at 0, 0.5, 1, 2 and 4 h after serum induction. The induction curves shown in Fig. 5 show a 2-fold induction in c-myc level for both phenotype classes of colon tumor cell lines, although the absolute c-myc level achieved for HCT116 cells was 2-fold higher than that achieved by FET cells, i.e., 5500 cpm versus 2800 cpm, respectively. Group
Influence c-myc human
of differentiation-inducing agents on c- myc levels Human colon tumor cell lines with well- and poorly-differentiated phenotypes were grown in the presence of TGF-P, RA, DMF or sodium butyrate for 5 days and examined for their cmyc levels. Comparisons of untreated controls with treated cell lines are shown in Table I. Only well-differentiated cell lines were generally responsive to TGF-/3 and retinoic acid with
Influence
of c-myc
I and
Group
by serum
III colon
tumor
of differentiation-inducing induction colon
in tumor
Group
I and
agents
on
Group
Ill
cell lines
The influence of TGF-0, RA, DMF and sodium butyrate on the induction of c-myc expression was tested by introducing the agents 24 h before serum stimulation, as described in Materials and Methods. A summary of the influence of each differentiation-inducing agent on the induction curve for HCT116 and FET cells is presented in Table II. Induction levels in HCT116 cells were inhibited by sodium butyrate, dimethylformamide and retinoic acid, but
Fig. 4. Photomicroscopy of HCT116 and FET cells grown in the presence and absence of 2.0 mM sodium butyrate. Cells were seeded in 6-well plates and media containing 2.0 sodium butyrate changed as described in Fig. 3. Plates A and B represent HCTl16 and FET cells grown in the presence of 2.0 mM sodium butyrate, respectively. Plates C and D represent control HCTl16 and FET cells grown 5 days in McCoy’s 5A medium containing 10% FBS. Photographs represent an original 240-fold magnification.
G c
102
Table 1. Influence of differentiation-inducing agents on natural c-myc protein levels. Following autoradiographic localization of the 62-kDa band on the nitrocellulose sheet, regions corresponding to the c-myc band were cut from the sheet and counted in a Beckman Gamma 4000 counter. In each case enough counts were collected to achieve a 98% confidence level. These values represent the averages determined for three separate experiments. Percent
Decrease
TGF-/3 Group I HCTl16 C RKO
DMF
Sodium
1.3 0 0
5 27 26
72 38 36
1.0 30 12
51 29 75
57 49 59
RA
1.6 3.0 1.0
Group III FET GE0 GLY
78 42 53
not by TGF-/3. The order of influence for this inhibition was sodium butyrate > dimethylformamide > retinoic acid. With the exception of retinoic acid there is a good correlation between the influence of these agents on c-myc constitutive levels (Table I) and their effects
1
2 Time
3
butyrate
upon c-myc induction (Table II). The inhibition of c-myc induction in FET cells showed that all differentiation-inducing agents, except retinoic acid, influenced c-myc levels. The order of inhibition for individual agents was TGF-fl > > dimethylformamide. sodium butyrate Again, the correlation between these data (Table II) and the influence of these agents on constitutive c-myc levels (Table I) is quite good.
I
4
(hr)
Fig. 5. Induction of c-myc in HCT116 and FET cells by serum replenishment following 96 h of serum starvation. Colon tumor cells were seeded in 75-cm* cell culture flasks, permitted to attach for 24 h and grown to 75% confluence. Upon reaching 75% confluence cells were starved for 96 h in McCoy’s 5A medium before serum replenishment with 10% FBS. Cells were removed at 0. 0.5, 1, 2 and 4 h after re-feeding. Crude nuclei were prepared, electrophoresis run on 10% polyacrylamide Laemmli gels [ 191 and immunoblotted onto nitrocellulose sheets. Nuclear c-myc levels were quantitated by measuring 1251-labeled protein G binding in a gamma counter.
ir
0.04 0.0
0.5
1.0
LO.0 1.5
2.0
Sodium Butymts (mM)
Fig. 6 Graph showing the correlation between nuclear cmyc level and percent of control growth as a function of sodium butyrate concentration. HCTl16 cells were plated in 6-well plates. After cell attachment, HCT116 cells were grown in the presence of 0.0, 0.5, 1.0, 1.5 and 2.0 mM sodium butyrate for 5 days. Cells were collected and analyzed for cell number by cell counting in a hemocytometer. The nuclear c-myc levels were measured as described in Fig. 2.
-
103 Table II.
of differentiation-inducing agents on the induction of c-myc in human colon tumor cell lines. The relative to untreated controls were calculated and reported as percentages. MIL values represent the counts determined at the maximum level of induction (MIL), HCTl16 and FET cells were treated with 10 ng/ml TGF-/3, 1.0 mM retinoic acid, 1% DMF or 2.0 mM sodium butyrate as described in Methods and Materials. Influence
percent decrease in c-myc induction
HCTl16
Treatment
Control TGF-6 Retinoic Acid DMF Sodium butyrate “The values reported
Correlation HTCl16
Percenta
MIL x lo-:’
Percent a
MIL x 10 - ’
100 100 67 57 33
2.9 2.9 2.8 2.9 0.6
100 38 95 67 43
2.8 0.7 0.7 0.3 0.8
for c-myc induction
of c-myc
level
with
are calculated
growth
rate in
cells
To better tween
FET
growth
understand the relationship berate and c-myc level in HCTl16
cells and their inhibition by sodium butyrate a dose-response study was performed. The concentration of sodium butyrate was increased in 1 mM increments from 0.0-4.0 mM and the number of cells and the c-myc level determined. A plot of percentage growth and ‘251-protein G binding to anti-c-myc antibody versus sodium butyrate dose is shown in Fig. 6. The curves showing a reduction in HCTll6 cell number and c-myc level show a similar downward trend with increasing sodium butyrate concentration. Discussion
Analysis of the c-myc levels in human colon tumor cell lines of defined biological phenotype has shown that there is an inverse correlation between the c-myc level and a cell’s differentiation phenotype. More differentiated cell lines that are members of the phenotype Group III have the lowest individual and collective c-myc levels. Poorly differentiated cell lines that have been shown to be more tumorigenic have the highest c-myc levels. The ratio calculated for the average level of c-myc expression is 1.7: 1. In this regard, the lower c-
as percentages
of that induction
seen for controls.
myc level in cells of more differentiated phenotype is consistent with the very similar results reported for HL-60 and U - 937 cells [9,25,32]. When Group I and Group III human colon tumor cell lines were analyzed to determine the influence of a variety of differentiation-inducing agents on their c-myc levels it was approached in two ways. The first involved an analysis of the influence of TGF-/3, RA, DMF and sodium butyrate on constitutive c-myc levels. It was found that the more differentiated Group III human colon tumor cell lines were the most sensitive. Nearly all of the Group III cell lines analyzed were sensitive to these agents. FET cells were the only exception, showing no response to retinoic acid. Poorly differentiated cell lines showed a more limited set of responses. Levels of c-myc protein in poorly differentiated colon tumor cell lines were only reduced following treatment with DMF or sodium butyrate, the influence of DMF in HCT116 cells being marginal. The second analysis involved the measurement of c-myc induction levels following serum re-feeding. With the exception of TGF-0 treatment of HCT116 cells. all c-myc inductions were inhibited to some extent by the other three differentiation inducing agents. The well-differentiated cell lines showed more sensitivity to TGF-P. sodium butyrate and DMF.
104
The issue of c-myc inducible level versus cmyc fold-induction ratio was also addressed in Table 11. Both the percent of control and maximum inducible level values were determined and compared. These results demonstrate a good correlation between the ranking of effects based upon the maximum inducible c-myc levels and that rank order determined for percentage values that are based upon a comparison with untreated control values. These results suggest that variation in c-myc level provides a good indication of the loss of colon tumor cell differentiated function. As such, c-myc levels may have a function in adenocarcinoma of the colon in addition to driving proliferation, i.e., progression to more tumorigenic and less differentiated phenotypes. It seems quite likely, therefore, that there are specific levels of c-myc that are required for defined levels of growth or tumorigenicity. This type of speculation is very reasonable when one considers the competency model for c-myc function in promoting or maintaining malignant transformation characteristics [23]. Finally, the relationship between actual c-myc protein levels measured in 1% DMFtreated HCT116 cells and the 90% reduction in c-myc mRNA level reported by Mulder and Brattain [Zl] illustrates several important features of this study. The first is a reiteration of the complicated nature of c-myc regulation. The second is the importance of measuring cmyc protein levels. It appears that to really understand the mechanisms used by differentiation-inducing agents and the predictability of their potential applications in clinical colon cancer, it is important to determine the extent to which they attenuate cellular oncogene product levels. The third is the better understanding that studies of this type will provide in realizing the importance of c-myc dose if c-myc is to be used as a growth and/or differentiation inhibition target.
Barbara Young in the culturing of cells. Support for these studies was provided by National Cooperative Drug Discovery Grant CA45967 awarded by the NC1 and by Bristol-Myers Squibb Company. References 1
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597 - 600.
Litwin. S., Keidan. R.D.,
(1988)
the c-myc oncogene
Noncorrelation in colorectal
Comis, R.L. and
of the expression of carcinoma
with recur-
rence of disease or patient survival. Proc. Natl. Acad. Sci. U.S.A., 11
48,
1350-
Erisman, M.D., Spandorfer,
1355.
Rothberg, P.G.,
Diehl. R.E., Morse, C.C.,
J.M. and Astrin, S.M.
(1985)
Deregulation
c-myc gene expression in human colon carcinoma accompanied
Acknowledgements
by amplification
gene. Mol. Cell Biol., 5, 196912
The authors would like to acknowledge the excellent technical assistance provided by Ms.
G., Bishop, J.M.,
McGrath,
Erisman, M.D., (1988) elevated cogene,
Scott, J.K.,
The c-myc protein
of the
1976.
Wald,
R.A. and Astrin. S.M.
is constitutively
levels in colorectal 2, 367-378.
or rearrangement
of
is not
carcinoma
expressed
cell lines.
at On-
105
Evans, G.I and Hancock, D.C. (1985) Studies on the interaction of the human c-myc protein with cell nuclei: p62‘-mYc as a member of a discrete subset of nuclear proteins. Cell, 43, 253-261.
24
14 Finley, G.G.. Schulz, W.T.. Hill. %A.. Geisler. J.R., Pipas, J.M. and Meister. A.I. (1989) Expression of the myc gene family in different stages of human colorectal cancer. Oncogene, 4. 963 - 971.
25
13
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23
Harm, S.R.. Abrams, H.D., Rohrschneider, L.R. and Eisenman, R.N. (1983) Proteins encoded by v-myc and cmyc oncogenes: Identification and localization in acute leukemia virus transformants and bursal lymphoma cell lines. Cell, 34. 789 - 798. Imaseki, H., Hayashi, H., Taira. M., ho, Y., Tabata, Y., Onoda, S., Isono. K. and Tatibana, M. (1989) Expression of c-myc oncogene in coloredal polyps as a biological marker for monitoring malignant potential. Cancer, 64, 704- 709. Kaczmarek, L., Hyland, J., Watt, R.. Rosenberg, M. and Baserga, R. (1985) Microinjected c-myc as a competence
26
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29
Laemmli. U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227, 680 - 685. Marks, M.E., Ziober. B.L. and Brattain, M.B. (1986) N,Nantidimethylformamide-induced synthesis of an fibronectin reactive protein in cultured human colon carcinoma cells. Cancer Res., 46, 5248-5258. Mulder. K.M. and Brattain, M.B. (1988) Alterations in c-
30
poorly differentiated and well differentiated human colon carcinoma cells. Mol. Endocrinol.. 1. 1215- 1222. Pledger, W.J.. Stiles, C.D., Antoniades, H.N. and Scher. C.D. (1978) An ordered sequence of events is required before BALB/c-3T3 cells become committed to DNA synthesis. Proc. Natl. Acad. Sci. U.S.A., 75, 2839 - 2843.
G.W. down
regulation of the c-myc gene by sodium butyrate in Burkitt’s lymphoma cells. EMBO J.. 6, 2959-2964. Reitsma, P.H., Rothberg. P.G., Astrin, S.M.. Trail, J., Bar-Shavit, Z., Hall, A., Teitelbaum, S.L. and Kahn, A.J. (1983) Regulation of myc gene expression in HL-60 leukemia cells by a vitamin D metabolite. Nature (London), 306, 492 - 494. Sachs, L. (1980) Constitutive uncoupling of pathways of gene expression that control growth and differentiation in myeloid leukemia: A model for the origin and progression of malignancy. Proc. Nab. Acad. Sci. U.S.A., 77, 6152-6156.
factor. Science, 228, 1313 - 1315. Kelley, K.. Cochran, 8.. Stiles, C. and Leder, P. (1984) The regulation of c-myc by growth signals. Curr. Top. Microbial. Immunol.. 113, 117- 126.
myc expression in relation to maturational status of human colon carcinoma cells. Int. J. Cancer, 42, 64- 70. Mulder. K.M. and Brattain, M.G. (1989) Effects of growth stimulatory factors on mitogenicity and c-myc expression in
Polack. A., Eick. D., Koch, E. and Bornkamm, (1987) Truncation does not abrogate transcriptional
31
32
33
Slamon, D.J.. DeKernion. J.B., Verma. I.M. and Cline, M.F. (1984) Expression of cellular oncogenes in human malignancies. Science, 224, 256 - 262. Steward, J., Evan, G.. Watson, J.. Sikora, K. (1986) Detection of the c-myc oncogene product in colonic polyps and carcinomas, Br. J. Cancer, 53, l-6. Taylor, C.W., Brattain, M.G. and Yeoman, L.C. (1984) Rate of growth and extent of differentiation reflected by cytoplasmic proteins and antigens of human colon tumor cell lines. Cancer Res., 44. 1200 - 1205. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76, 4350 -4354. Wan, C.-W. McKnight, M.K., Brattain, D.E., Brattain, M. and Yeoman, L.C. (1988) Growth requirements and receptor levels for epidermai growth factor in human colon carcinoma cell lines. Cancer Lett., 43, 139 - 143. Westin. E.H., Wong-Staal. F., Gelmann. E.P., Dalla Favera. R., Papas, T.S.. Lautenberger, J.A., Eva, A., Reddy, E.P., Tronick. S.R., Aaronson, S.A. and Gallo. R.C. (1982) Expression of celjutar homologues of retroviral one genes in human hematopoietic cells. Proc. Natl. Acad. Sci. U.S.A., 79, 2490-2494. Yarden, A. and Kimichi, A. (1986) Tumor necrosis factor reduces c-myc expression and cooperates with interferongamma in HeLa cells. Science, 234, 1419- 1421.
95 - 105
95
Elsevier Scientific Publishers Ireland Ltd.
Sensitivity of nuclear c-myc levels and induction to differentiation-inducing agents in human colon tumor cell lines C.W. Taylora, Y.S. Kimb, K.E. Childress-Fields” of Pharmacology.
‘Department Veterans
Administration
(Received
21 June
(Accepted
17 October
Baylor
Medical
College
Center.
of Medicine.
4150
Clement
and L.C. Yeomana
Houston.
Street,
San
TX
Six human colon tumor cell lines were analyzed for their constitutiue leuels of the c-myc protein. The nuclear proto-oncogene, c-myc, was detected as an expressed product in all of the human colon tumor cell lines analyzed.
levels
poorly differentiated cell lines HCTl16, and C showed c-myc leuels that aueraggreater
counterparts, c-myc levels were
oic acid,
colon
their
sodium
butyrate
and
TGF-/3,
of sensitiuity and resistance c-myc leuels were reduced cell phenotypes
formamide
treated
or sodium
with
dimethyl-
Only the well-
butyrate.
Only one of the human
lines
(GEO)
responded
Correspondence Abbreuiotions: mamide;
DOC,
colon
to
tumor
retinoic
cell
acid.
In-
to’ Lynn C. Yeoman. CPM.
counts per minute;
deoxycholate;
EGF.
FBS. fetal bovine serum; HCT, phenylmethanesulfonyl radioimmunoassay; transforming
growth
SDS,
sodium
factor-beta
0
1992
Printed and Published in Ireland
DMF.
epidermal
human
fluoride;
ethylaminomethane.
0304.3835/92/$05.00
Lab..
levels of c-myc protein were found to correlate well with greater growth rates and with poor differentiation class. Similarly, a parallel sensitivity to down-regulation of c-myc and
attenuation
curues for inducers observed in growth
of
c-myc
induction
of differentiation sensitive human
were colon
Keywords: c-myc; colon; oncogene; oncogene, induction and differentiation
protoagents
tumor
cell lines.
RA.
colon tumor:
dodecyl and
dimethylforgrowth factor:
retinoic
PMSF.
acid:
sulfate:
TRIS.
Introduction
distinct
emerged. in all the
differentiated human colon tumor cell lines were responsiue to transforming growth factorfl.
Research
(U.S.A.)
well-differentiated
in the presence of inducers of i.e., dimethylformamide, retin-
analyzed
patterns Nuclear
than
i.e., GEO, CBS and FET. When and responses to serum induction
differentiation,
‘Gastrointestinal 94121
1991)
creased
ed Z-fold
and CA
1991)
Summary
The MO
77030
Francisco,
RIA.
TGF-8.
trihydroxy-
In previous studies we have used immunological probes to analyze the cellular antigens expressed by a group of highly characterized human colon tumor cell lines [29]. These human colon tumor cell lines have been divided into three groups based upon their biological characteristics. Group 1 cell lines produce poorly differentiated tumors in athymic nude mice, grow we11 in soft agarose and secrete low levels of high molecular weight mucins into their media. Group III cell lines produce well differentiated zenografts in athymic nude mice, do not grow as efficiently in soft agarose, but secrete high levels of carcinoembryonic antigen into their culture medium. Group II cell lines fall between Groups I and III in their biological properties. The ex-
Elsevier Scientific Publishers Ireland Ltd
96
pression of specific tumor associated antigens revealed patterns of cytosolic antigen expression that correlated with the differentiation class of the individual cell lines [29]. In studies designed to examine the epiderma1 growth factor sensitivity of colon tumor cells we have shown that their growth factor to exogenous EGF’ and their responses numbers of cell surface EGF-receptors correlate with cell grouping [31]. More differentiated cell lines express more cell membrane associated EGF-receptor and show a substantial increase in growth response to exogenous EGF, whereas poorly differentiated colon tumor cell lines are insensitive to exogenous growth factor and exhibit low levels of cell membrane associated EGF-receptor. There is a good correlation between tumor associated antigen expression, growth factor receptor expression and cell phenotype in our cell line model system for human colon cancer [4,5]. Because of the reported induction of c-myc transcripts in response to growth signals [18] we have evaluated the levels of c-myc expression in this series of well characterized human colon tumor cell lines. The c-myc protein is a 439-amino acid phosphoprotein of 51 kDa molecular weight that has been shown to migrate on SDS containing polyacrylamide gels as a 62/64 kDa band [1,2,8,15]. Using various inducers of differentiation in the study of malignant hematopoietic cells the c-myc nuclear proto-oncogene has been shown to immortalize and/or transform cells as a primary oncogene [26]. It mediates signal transduction in the cell nucleus through its DNA-binding activity [8]. The net effect of c-myc on transformation and tumor progression is thought to occur as a result of proliferative responses to c-myc expression. This is thought to cause an induction of replication competence during the cell cycle [17]. Studies on the effects of inducers of differentiation in malignant hematopoietic cells suggest that c-myc expression is decreased during the induction of differentiation [9,25,32]. The importance of understanding c-myc expression at the protein level is illustrated by the
complex manner in which c-myc levels are regulated. The level of c-myc expression has been shown to be regulated at the point of transcription initiation, transcriptional elongation, mRNA stability and protein translation
171. There appears to be a strong correlation between tumorigenicity and cell grouping in the human colon tumor lines we have studied. Furthermore, reports from other laboratories have described correlations between c-myc level, malignancy and tumor size in clinical studies on human colorectal and gastric cancer [6,11,16,27,28]. We became interested, therefore, in determining whether c-myc level is related to cell grouping in this set of characterized human colon tumor cell lines. Finley et al. [14] found modest c-myc overexpression in approximately two-thirds of the colorectal carcinoma cases that they examined. These reports are consistent with those made earlier by Erisman et al. [lo] who showed that expression of the c-myc gene at both the mRNA and protein level occurs constitutively and is 8- to 37-fold higher than levels of c-myc protein in normal mucosal cells. The predictive use of c-myc levels in clinical specimens, however, has not proved useful as a correlate for clinical outcome in malignancies of the colon [lo]. We have hypothesized that analyses performed in a set of defined colon cell lines that exhibit a wide range of biological characteristics could be very useful. It has been shown that c-myc transcripts can be downregulated by a variety of different agents in a large number of cell types [24,33]. By using differentiation-inducing chemicals and peptides, the influence of these agents on c-myc levels in colon cell lines of defined differentiation phenotype could be determined. The use of colon tumor cell lines avoids the clonal heterogeneity associated with the study of solid tumors. Similarly, the influence of invading cells can be eliminated. Because of the complex nature of the regulation of c-myc expression, determining the protein level of the c-myc proto-oncogene product is probably the only reliable indicator of c-
myc function in growth and differentiation [26]. We have analyzed the constitutive level of c-myc expression in six colon cell lines. Three were selected from each of the two most divergent phenotype classes [4,5]. By examining constitutive c-myc levels, determining cmyc enhancement following serum induction and analyzing the effects of differentiationinducing agents on c-myc levels and cell growth rates we have established a clear relationship between c-myc level and cell phenotype in human colon tumor cells. Materials and Methods Tissue culture and cell fines The establishment and characterization of a series of human colon tumor cell lines that were derived from primary colon tumors have been described previously [3]. These cell lines have been divided into three groups based upon their in vitro biological characteristics and tumorigenicity in athymic nude mice [4,5]. These studies were performed on cells grown: (i) in supplemented McCoy’s 5A medium containing 10 percent FBS, (ii) starved for 4 days in the presence of supplemented McCoy’s 5A medium and (iii) re-fed with 10% FBS in supplemented McCoy’s 5A medium. Mitogenesis assay Cells were seeded at densities of 20 000 cells per 35-mm well in McCoy’s 5A medium containing 10% FBS. Cells were grown to 75% confluence and starved for 5 days. Cells were given a l-h pulse with [“Hlthymidine 50 Ci/mmole; ICN (100 ~1; 25 &i; Radiochemicals, Irvine, CA) at 37OC [22] after 0, 1, 2, 3, 4 and 5 days of starvation. Thymidine-containing medium was removed and the cells were washed with 1.0 ml of plain McCoy’s 5A medium. Cell membranes were lysed by incubation for 10 min with 10% trichloroacetic acid at 4OC and washed once with trichloroacetic acid. The contents of each well were removed with 0.5 ml of 0.2 M NaOH containing 40 pg/ml calf thymus DNA. Samples were analyzed for [3H]thymidine-
labeled DNA by counting in a Beckman ~~7500 liquid scintillation counter (Beckman Instruments; Palo Alto, CA). Serum starvation and induction of c-myc Cell lines were plated and grown from 70-80% confluence in supplemented McCoy’s 5A medium containing 10% fetal bovine serum (10F). Cells were subsequently starved by incubation for 96 h in McCoy’s 5A medium. Cells were fed again at time zero (T = 0) with media containing 10% fetal bovine serum in the presence or absence of TGF-P (10 ng/ml), retinoic acid (1 .O PM), dimethylformamide (1.0%) or sodium butyrate (2 mM). Preparation of whole cell Iysates and crude nuclei Cells were detached by scraping and washing with phosphate buffered saline containing 1 .O mM phenylmethanesulfonyl fluoride (PMSF). Washed cells were suspended in a modified Laemmli sample buffer [19] containing 1% SDS, 10% glycerol, 0.2 M Tris-HCI (pH 6.8), 1.0 mM PMSF, 0.1 mM leupeptin and 0.1 mM aprotinin. Cells were repeatedly dispersed and homogenized by sequential passage through 18-, 21. and 25-gauge syringe needles. Homogenization was repeated until the solution was no longer viscous. Crude nuclei were prepared by the low salt procedure of Evans and Hancock [13]. Cells were washed with PBS and suspended in low salt lysis buffer containing 20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 mM KCI, 5 mM MgC&, 0.5% Nonidet P-40, 0.1% DOC, 1.0 mM PMSF, 0.1 mM leupeptin and 0.1 mM aprotinin. After mixing by repeated pipetting, cells were lysed for 5 min at 4OC with intermittent mixing. Nuclei were collected by centrifugation at 1000 X g for 5 min and washed in lysis buffer before suspension in a modified Laemmli sample buffer [19]. Polyacrylamide gel electrophoresis and immunoblotting Poiyacrylamide gel electrophoresis was per-
98
formed as described by Laemmli [ 191. Precisely 75 pg of protein lysate, containing bromophenol blue and 2-mercaptoethanol, were loaded into each sample well of a 10% polyacrylamide SDS slab gel. Electrophoresis was performed in a Bio-Rad Protean Cell (BioRad; Richmond, CA) and continued at 150 V until the bromophenol blue dye front was within 0.5 cm of the end of the gel (approximately 2.5 -3.0 h). Proteins were electrophoretically transferred to nitrocellulose paper in a Bio-Rad Trans-Blot Cell at a current of 250 mA for 4 h as described by Towbin et al. [30]. Prior to immunoblotting the nitrocellulose sheets were blocked with a PBS solution containing 3% RIA grade BSA (Sigma, St. Louis, MO) and 0.5% Tween-20. Immunodetection was achieved using a pan reactive anti-c-myc antibody (sheep IgG) obtained from Cambridge Research Biochemicals (Valley Stream, NY). The primary antibody was used at a 1:lOO dilution for 2 h at 25OC; the rabbit anti-sheep second antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1:500 dilution for 2 h at 25OC and ‘251-protein G (DuPont Company; Boston, MA) at a concentration of 1 x lo5 cpm/ml for 2 h at 25OC. All antibody and protein G solutions were diluted in a PBS solution containing 1% RIA grade BSA and 0.5% Tween-20. All blocking and immunoblot steps were performed with gentle mixing (75 rev./min) on a Gyratory Shaker (New BrunsEdison, NJ) wick Scientific Instruments, Autoradiographic patterns were obtained by imexposing developed, nitrocellulose munoblots to Kodak X-Omat AR-5 film with Du Pont Chronex intensifier screens (Du Pont Company; Boston, MA) for 1 - 4 h. Quantitative
comparison
of c-myc
levels
In preparation for quantitation the corners of immunoblotted nitrocellulose sheets were spotted with r4C-labeled radioactive ink to permit the precise alignment of Xerox transparency sheets of autoradiograms with the nitrocellulose blots. Following alignment of the xerographic pattern with the nitrocellulose
sheet, the 62 kDa band was outlined, cut out and counted in a Beckman 4000 gamma counter (Beckman Instruments, Palo Alto, CA). Samples were counted for 10 min or 100 000 counts, whichever came first and corrected for background. Background values were obtained for several pieces of nitrocellulose of comparable area cut from regions of the nitrocellulose sheet that were devoid of visually detectable autoradiographic density and averaged. Results Specificity
of the c-myc
molecular
weight
tein
and
human
constitutiue colon
polyclonal
behavior
tumor
of the
c-myc
protein
antibody, c-myc
pro-
levels
in
cell lines
To establish that the immunoreactive bands detected in whole cell extracts and crude nuclear fractions prepared from human colon tumor cell lines were authentic c-myc gene products, immunoblots were performed using both c-myc reactive and antibody that had been preabsorbed with a lo-fold excess of immunizing peptide. The immunoblot pattern obtained when nuclear fractions from HCTl16, RKO, C, FET, GE0 and GLY cells were immunoblotted is shown in Fig. 1. Preabsorption with immunizing peptide eliminated the detection of the authentic 62-kDa c-myc band (data not shown). A molecular weight of 62 kDa was calculated for the single immunoreactive band. This value was based upon the migration of the immunoreactive band relative to protein standards of known molecular weight and agrees well with the reported molecular weight behavior of c-myc [1,2,8,15] Measuring the level of ‘251-protein G binding to the respective lanes shown in Fig. 1 revealed that c-myc protein levels were uniformly higher in poorly differentiated colon tumor cell cultures (Group III cell lines: HCT116, RKO and C) when compared to well-differentiated cell lines (Group I cell lines: FET, GE0 and GLY). The average c-myc level (14 400 cpm) in a poorly differentiated colon tumor cell line was 1.7 times higher than
99
_._
7 0
Y
62k
-45
E 0” cl .g
1.5~-
1.0-m
b k
-31
&
0.5-m
3 x2 0.01
RKO
HCT116
C
GE0
GLY
FEX
HCT Cell Line
HCT.116 RKO -II
Group I
C
FET
GE0
GLY
Group III
Fig. 1. Quantitative immunoblot analysis of human colon tumor cell nuclear extracts with anti-c-myc polyclonal antibody. Nuclear fractions were prepared from Group I (HCT116, RKO and C) cells and Group III (FET, GE0 and GLY) cells and 75 pg loads subjected to electrophoresis on 10% Laemmli gels [19]. Protein was transferred to nitrocellulose sheets according to the method of Towbin [30] and immunoreactivity probed with polyclonal anti-c-myc antibody. Immunoreactive bands were detected using 1251-labeled protein G and autoradiography. The c-myc protein band is marked with an arrow at 62 kDa.
the corresponding average level determined for well differentiated colon cells (8500 cpm). The data shown in the bar graph in Fig. 2 indicate that the range of ‘251-protein G counts associated with the c-myc band extended from 7500 cpm for FET cells to 18 000 cpm for RKO cells. Influence tumor
of sodium butyrate on human colon
cell growth
Before the influence of sodium butyrate concentration on c-myc levels could be addressed, it was necessary to determine the influence of butyrate dose on colon tumor cell growth. Both HCT116 cells and FET cells were sub-
Fig. 2. Comparison of natural c-myc levels in six human colon tumor cell lines. Crude nuclei were prepared from RKO, HCT116, C, GEO. GLY and FET cells and subjected to polyacrylamide gel electrophoresis and immunoblotting. Binding of ‘251-labeled protein G to sheep polyclonal anti-c-myc antibody on nitrocellulose sheets was determined by cutting autoradiograph localized bands from the nitrocellulose sheet and counting in a gamma counter.
jetted to growth for 5 days in the presence of sodium butyrate concentrations ranging from 0 - 4 mM. Additions of sodium butyrate were made on days 1 and 3 when culture media was changed. The different percentages of inhibition of colon tumor cell line growth by sodium butyrate are shown in Fig. 3. Both HCT116 (poorly-differentiated) and FET (well-differentiated) cells were inhibited by sodium butyrate. IC50 values of 0.34 mM and 0.80 mM were calculated for HCT116 and FET respectively. The appearance of cells, HCT116 and FET cells after being grown in the presence of 2 mM sodium butyrate for 5 days is shown in Fig. 4. Because there was a substantial reduction in the number of cells counted in cultures grown in the presence of sodium butyrate, the question of butyrate toxicity was addressed. Staining of 2 mM sodium butyrate-treated and -untreated control cultures with Trypan Blue did not reveal a significant difference in cell viability (I 2 - 5% dye uptake) between butyrate-treated and control cell cultures (data not shown).
c-myc levels reduced from 42 - 78% of control for TGF-fl and 12 -30% of control for retinoic acid (when the influence of RA on FET cells is excluded). Cellular responses to DMF were more prevalent, i.e., DMF caused decreases ranging from 5 - 27% in poorlydifferentiated cell lines and 29 - 75% in welldifferentiated cells. Sodium butyrate, on the other hand, showed a strong effect upon cmyc levels in both poor- and well-differentiated human colon tumor cell lines. Poorly-differentiated cell lines showed 36 - 72% reductions in c-myc level while well-differentiated lines ranged from 49-59%. Induction
0
1 Sodium
2 Butyrate
3
4
(mM)
Fig. 3. Inhibition of HCT116 and FET cell growth by treatment with different concentrations of sodium butyrate. Human colon tumor cell lines were seeded at a density of 1 x lo6 cells per well in 6-well plates and permitted to attach for 24 h. Media were changed and replaced with fresh media containing 0.5, 1.0, 1.5, 2.0 and 4.0 mM sodium butyrate. Fresh media containing equivalent concentrations of sodium butyrate were added again on day 3 and cells counted on day 5. Results are presented as percentage inhibition with 100% based upon a 0.0 mM sodium butyrate control.
It has already been shown that DMF inhibits growth in a variety of human colon tumor cell lines [ZO], TGF-0 effects are limited to welldifferentiated cell line members of Group 111 and retinoic acid has little or no influence on colon tumor cell line growth [21].
refeeding in cell lines To determine the c-myc responses that accompany serum re-feeding, HCTl16 cells from Group I and FET cells from Group III were seeded and grown to 75% confluence in the presence of 10% FBS before starving for 4 days. Measurement of the fall in [3H]thymidine incorporation into DNA was used to establish the appropriate length of the starvation period. The c-myc levels were measured at 0, 0.5, 1, 2 and 4 h after serum induction. The induction curves shown in Fig. 5 show a 2-fold induction in c-myc level for both phenotype classes of colon tumor cell lines, although the absolute c-myc level achieved for HCT116 cells was 2-fold higher than that achieved by FET cells, i.e., 5500 cpm versus 2800 cpm, respectively. Group
Influence c-myc human
of differentiation-inducing agents on c- myc levels Human colon tumor cell lines with well- and poorly-differentiated phenotypes were grown in the presence of TGF-P, RA, DMF or sodium butyrate for 5 days and examined for their cmyc levels. Comparisons of untreated controls with treated cell lines are shown in Table I. Only well-differentiated cell lines were generally responsive to TGF-/3 and retinoic acid with
Influence
of c-myc
I and
Group
by serum
III colon
tumor
of differentiation-inducing induction colon
in tumor
Group
I and
agents
on
Group
Ill
cell lines
The influence of TGF-0, RA, DMF and sodium butyrate on the induction of c-myc expression was tested by introducing the agents 24 h before serum stimulation, as described in Materials and Methods. A summary of the influence of each differentiation-inducing agent on the induction curve for HCT116 and FET cells is presented in Table II. Induction levels in HCT116 cells were inhibited by sodium butyrate, dimethylformamide and retinoic acid, but
Fig. 4. Photomicroscopy of HCT116 and FET cells grown in the presence and absence of 2.0 mM sodium butyrate. Cells were seeded in 6-well plates and media containing 2.0 sodium butyrate changed as described in Fig. 3. Plates A and B represent HCTl16 and FET cells grown in the presence of 2.0 mM sodium butyrate, respectively. Plates C and D represent control HCTl16 and FET cells grown 5 days in McCoy’s 5A medium containing 10% FBS. Photographs represent an original 240-fold magnification.
G c
102
Table 1. Influence of differentiation-inducing agents on natural c-myc protein levels. Following autoradiographic localization of the 62-kDa band on the nitrocellulose sheet, regions corresponding to the c-myc band were cut from the sheet and counted in a Beckman Gamma 4000 counter. In each case enough counts were collected to achieve a 98% confidence level. These values represent the averages determined for three separate experiments. Percent
Decrease
TGF-/3 Group I HCTl16 C RKO
DMF
Sodium
1.3 0 0
5 27 26
72 38 36
1.0 30 12
51 29 75
57 49 59
RA
1.6 3.0 1.0
Group III FET GE0 GLY
78 42 53
not by TGF-/3. The order of influence for this inhibition was sodium butyrate > dimethylformamide > retinoic acid. With the exception of retinoic acid there is a good correlation between the influence of these agents on c-myc constitutive levels (Table I) and their effects
1
2 Time
3
butyrate
upon c-myc induction (Table II). The inhibition of c-myc induction in FET cells showed that all differentiation-inducing agents, except retinoic acid, influenced c-myc levels. The order of inhibition for individual agents was TGF-fl > > dimethylformamide. sodium butyrate Again, the correlation between these data (Table II) and the influence of these agents on constitutive c-myc levels (Table I) is quite good.
I
4
(hr)
Fig. 5. Induction of c-myc in HCT116 and FET cells by serum replenishment following 96 h of serum starvation. Colon tumor cells were seeded in 75-cm* cell culture flasks, permitted to attach for 24 h and grown to 75% confluence. Upon reaching 75% confluence cells were starved for 96 h in McCoy’s 5A medium before serum replenishment with 10% FBS. Cells were removed at 0. 0.5, 1, 2 and 4 h after re-feeding. Crude nuclei were prepared, electrophoresis run on 10% polyacrylamide Laemmli gels [ 191 and immunoblotted onto nitrocellulose sheets. Nuclear c-myc levels were quantitated by measuring 1251-labeled protein G binding in a gamma counter.
ir
0.04 0.0
0.5
1.0
LO.0 1.5
2.0
Sodium Butymts (mM)
Fig. 6 Graph showing the correlation between nuclear cmyc level and percent of control growth as a function of sodium butyrate concentration. HCTl16 cells were plated in 6-well plates. After cell attachment, HCT116 cells were grown in the presence of 0.0, 0.5, 1.0, 1.5 and 2.0 mM sodium butyrate for 5 days. Cells were collected and analyzed for cell number by cell counting in a hemocytometer. The nuclear c-myc levels were measured as described in Fig. 2.
-
103 Table II.
of differentiation-inducing agents on the induction of c-myc in human colon tumor cell lines. The relative to untreated controls were calculated and reported as percentages. MIL values represent the counts determined at the maximum level of induction (MIL), HCTl16 and FET cells were treated with 10 ng/ml TGF-/3, 1.0 mM retinoic acid, 1% DMF or 2.0 mM sodium butyrate as described in Methods and Materials. Influence
percent decrease in c-myc induction
HCTl16
Treatment
Control TGF-6 Retinoic Acid DMF Sodium butyrate “The values reported
Correlation HTCl16
Percenta
MIL x lo-:’
Percent a
MIL x 10 - ’
100 100 67 57 33
2.9 2.9 2.8 2.9 0.6
100 38 95 67 43
2.8 0.7 0.7 0.3 0.8
for c-myc induction
of c-myc
level
with
are calculated
growth
rate in
cells
To better tween
FET
growth
understand the relationship berate and c-myc level in HCTl16
cells and their inhibition by sodium butyrate a dose-response study was performed. The concentration of sodium butyrate was increased in 1 mM increments from 0.0-4.0 mM and the number of cells and the c-myc level determined. A plot of percentage growth and ‘251-protein G binding to anti-c-myc antibody versus sodium butyrate dose is shown in Fig. 6. The curves showing a reduction in HCTll6 cell number and c-myc level show a similar downward trend with increasing sodium butyrate concentration. Discussion
Analysis of the c-myc levels in human colon tumor cell lines of defined biological phenotype has shown that there is an inverse correlation between the c-myc level and a cell’s differentiation phenotype. More differentiated cell lines that are members of the phenotype Group III have the lowest individual and collective c-myc levels. Poorly differentiated cell lines that have been shown to be more tumorigenic have the highest c-myc levels. The ratio calculated for the average level of c-myc expression is 1.7: 1. In this regard, the lower c-
as percentages
of that induction
seen for controls.
myc level in cells of more differentiated phenotype is consistent with the very similar results reported for HL-60 and U - 937 cells [9,25,32]. When Group I and Group III human colon tumor cell lines were analyzed to determine the influence of a variety of differentiation-inducing agents on their c-myc levels it was approached in two ways. The first involved an analysis of the influence of TGF-/3, RA, DMF and sodium butyrate on constitutive c-myc levels. It was found that the more differentiated Group III human colon tumor cell lines were the most sensitive. Nearly all of the Group III cell lines analyzed were sensitive to these agents. FET cells were the only exception, showing no response to retinoic acid. Poorly differentiated cell lines showed a more limited set of responses. Levels of c-myc protein in poorly differentiated colon tumor cell lines were only reduced following treatment with DMF or sodium butyrate, the influence of DMF in HCT116 cells being marginal. The second analysis involved the measurement of c-myc induction levels following serum re-feeding. With the exception of TGF-0 treatment of HCT116 cells. all c-myc inductions were inhibited to some extent by the other three differentiation inducing agents. The well-differentiated cell lines showed more sensitivity to TGF-P. sodium butyrate and DMF.
104
The issue of c-myc inducible level versus cmyc fold-induction ratio was also addressed in Table 11. Both the percent of control and maximum inducible level values were determined and compared. These results demonstrate a good correlation between the ranking of effects based upon the maximum inducible c-myc levels and that rank order determined for percentage values that are based upon a comparison with untreated control values. These results suggest that variation in c-myc level provides a good indication of the loss of colon tumor cell differentiated function. As such, c-myc levels may have a function in adenocarcinoma of the colon in addition to driving proliferation, i.e., progression to more tumorigenic and less differentiated phenotypes. It seems quite likely, therefore, that there are specific levels of c-myc that are required for defined levels of growth or tumorigenicity. This type of speculation is very reasonable when one considers the competency model for c-myc function in promoting or maintaining malignant transformation characteristics [23]. Finally, the relationship between actual c-myc protein levels measured in 1% DMFtreated HCT116 cells and the 90% reduction in c-myc mRNA level reported by Mulder and Brattain [Zl] illustrates several important features of this study. The first is a reiteration of the complicated nature of c-myc regulation. The second is the importance of measuring cmyc protein levels. It appears that to really understand the mechanisms used by differentiation-inducing agents and the predictability of their potential applications in clinical colon cancer, it is important to determine the extent to which they attenuate cellular oncogene product levels. The third is the better understanding that studies of this type will provide in realizing the importance of c-myc dose if c-myc is to be used as a growth and/or differentiation inhibition target.
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