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Retinoids Differentially Regulate the Proliferation of Colon Cancer Cell Lines
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
233, 321–329 (1997)
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Retinoids Differentially Regulate the Proliferation of Colon Cancer Cell Lines LaMonica V. Stewart and Mary L. Thomas1 Department of Pharmacology and Toxicology, Route J-31, University of Texas Medical Branch, Galveston, Texas 77555-1031
In this study, the proliferative effects of retinoids were examined in the MC-26 and LoVo colon adenocarcinoma cell lines. The proliferation of the LoVo cell line was not altered in the presence of the retinoids all trans-retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA). Both retinoids, however, stimulated the growth, as measured by cell proliferation, of MC-26 cells. atRA and 9-cis-RA were equipotent in increasing MC-26 cell proliferation, suggesting that the growth stimulation is mediated by one or more retinoic acid receptors (RARs). To determine the RAR which might be responsible for this growth stimulatory effect, we characterized the RAR subtypes which were present in both cell lines. mRNA for the RARa, RARb, and RARg were detected in the MC-26 cell. Of the RARs present in MC-26 cells, the RARa does not mediate the growth stimulatory effects of retinoids, for a selective RARa antagonist was unable to prevent the retinoidinduced increase in MC-26 cell growth. RARa, RARb, and RARg mRNA are also expressed in the LoVo cell line; the lack of growth-stimulation by retinoids in LoVo cells, therefore, does not seem to be due to the absence of RARs. The results obtained in these experiments demonstrate that the growth response elicited by retinoids can vary between colon cancer cells and that the differences in response may not be solely determined by the RAR subtypes which are expressed in a colon cancer cell line. q 1997 Academic Press
INTRODUCTION
One class of compounds which has been shown to play a key role in the regulation of cell growth is the retinoids. Retinoids are metabolites of vitamin A, a nutrient derived from carotenoids and retinyl esters present in the diet [1]. The biologically active retinoids, which include all trans-retinoic acid (atRA) and 9-cisretinoic acid (9-cis-RA), are believed to alter cell function via interactions with two types of receptors belonging to the steroid hormone receptor superfamily: the 1 To whom correspondence and reprint requests should be addressed. Fax: (409) 772-9642. E-mail: [email protected].
retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). In vitro experiments have demonstrated that retinoids inhibit the proliferation of several types of tumor cells grown in culture, including cell lines derived from teratocarcinomas, human myelocytic leukemias, breast tumors, and tumors of the prostate [2– 5]. The growth inhibitory effects of retinoids are also evident in vivo. For example, the incidence of chemically induced mammary tumors in rodents was decreased in animals treated with either atRA or 9-cisRA [6, 7]. Because retinoids are successful in preventing tumor development in vivo and tumor cell growth in vitro, analogs of the retinoids are presently being developed for use in humans as an anticancer therapy [3, 8]. The effects of retinoids on the growth and development of colon cancer have not been extensively examined. Conflicting data have been obtained from in vivo experiments studying the effects of retinoids on the development of chemically induced colon tumors in rats [9–12]. There are also few reports that detail the effects of retinoids on colon cancer cells in culture. It is therefore difficult to determine whether the inhibitory growth effects of retinoids produced in other types of tumors will also occur in colon cancer cells and tumors. To gain a better understanding of how retinoids might affect colon cancer cells, we examined the ability of biologically active retinoids to modulate the proliferation of two colon cancer cell lines, MC-26 and LoVo cells. We have previously demonstrated MC-26 cells can be growth-stimulated by physiological concentrations of the steroid 17-b-estradiol (E2) while LoVo cells are not [13, 14]. The proliferation of LoVo cells can also be modulated by compounds acting via steroid hormone receptors, for the growth of these cells has been shown to be inhibited by the sterol 1,25-dihydroxyvitamin D3 [15]. The results obtained in the present study reveal that different proliferative responses are produced by retinoids in these two colon cancer cell lines. MATERIALS AND METHODS Cell culture. MC-26 cells, a mouse colon adenocarcinoma derived from a 1,2-dimethylhydrazine-induced tumor [16], were obtained
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from the Department of Surgery at the University of Texas Medical Branch (UTMB). They were maintained in phenol red-free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% fetal bovine serum (FBS) (Atlanta Biological), 10 U/ml penicillin, and 0.01 mg/ml streptomycin in T-75 culture flasks (Corning). Cell passages between 30 and 40 were used in all experiments. The medium was changed every 2 days. LoVo cells, a human colon adenocarcinoma derived from a metastatic tumor nodule from a 56-year-old Caucasian male, were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were maintained in Hams F-12 medium supplemented with 10% FBS in T-75 culture flasks. Cells between passage 30 and 40 were used in all experiments. The medium was changed every 3 days. All cultured cells were kept at 377C in a 95% air/5% CO2 environment. They were subcultured weekly 1:10. Cell proliferation experiments. LoVo and MC-26 cells were plated at a density of 8,000–10,000 cells/well in 24-well culture plates and allowed to attach for 24 h. The medium was then changed (Day ‘‘0’’) to DMEM supplemented with 10% NuSerum, 10 U/ml penicillin, and 0.01 mg/ml streptomycin, and either retinoids or other steroid hormones dissolved in absolute ethanol were added to the medium. The final ethanol concentration in the wells was less than 0.1%. Control cells received an equal concentration of ethanol vehicle. To prevent degradation of the light-sensitive retinoids, drugs were added to the culture medium under subdued light. The medium was changed and fresh drug was added every 2 days. On Day 4, the cells were washed two times with phosphate-buffered saline and lysed by freeze-thawing the cells two times at 0707C. The lysates were further dispersed by placing them in a sonicating bath for 15 min. The amount of DNA in each well was then measured using the fluorescent dye, Hoechst No. 33258 (Sigma). An excitation wavelength of 356 nm and an emission wavelength of 458 nm was used to measure fluorescence [17]. In experiments involving the RARa antagonist, Ro 41-5253, the cells were pretreated for approximately 2 min with either RARa antagonist or dimethylsulfoxide (DMSO) vehicle. The retinoids or ethanol vehicle were then added to the wells. The final concentration of DMSO in the culture wells did not exceed 0.1%. The antagonist remained in the culture system for the duration of the experiment and was reapplied to the cultures whenever the medium was changed. Reverse transcription-polymerase chain reaction detection of retinoic acid receptor mRNAs. A GeneAmp Thermocycler (model 9600, Perkin–Elmer–Cetus) was employed for the reverse transcription and polymerase chain reactions. A computer algorithm [18] was used to select the sense and antisense oligonucleotide primers used in each experiment. The reverse transcription reaction was conducted in the presence of 0.5 mg total cellular RNA, AMV reverse transcriptase, and the antisense primer (0.75 mM) in a final volume of 10 ml for 30 min at 427C. The reverse transcriptase next was inactivated by heating at 957C for 5 min. PCR reagents, Taq DNA polymerase, and the sense primer (0.15 mM) were then added to a final volume of 50 ml, and 35 cycles of PCR amplification were performed (5 cycles at 947C for 15 s, 657C for 20 s, 727C for 15 s; 30 cycles at 947C for 15 s, 557C for 20 s, 727C for 15 s). The amplified products were analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide. A 306-bp fragment of the RARa mRNA was selected for amplification based on the published sequence for the mouse RARa cDNA [19]. Sense (5*-CGAACAACAGCTCAGAACAACG-3*) and antisense (5*-AAGGCAAAGACCAAGTCGG-3*) primers were chosen to amplify this fragment, which encodes a portion of the RARa ligand binding region common to all of the reported RARa isoforms. When compared to the published human RARa sequence, the sense primer is 100% homologous to the human RARa sequence, and there is
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only one base pair mismatch between the antisense primer and the human sequence. From the published mouse RARb2 mRNA sequence [20], both sense (5*-GAGCCTTCAAAGCAGGAATGC-3*) and antisense (5*TGAACACAAGGTCAGTCAGAGG-3*) primers were chosen to amplify a 430-bp region of RARb mRNA. This portion of RARb mRNA includes parts of the hinge and ligand binding regions and is present in all of the reported mouse RARb mRNA isoforms. A second set of sense (5*-GAAGCAAGAATGCACAGAGAGC-3*) and antisense (5*GATGGTCAAGCCAGTGAAACC-3*) primers was used to detect the presence of RARb in the human LoVo cell line. Based on the published human RARb cDNA sequence [21, 22], these primers were designed to amplify a 253-bp region of the hinge and ligand binding domains that is common to all of the reported human RARb isoforms. A 358-bp section of the RARg was chosen for amplification based on the published mouse RARg mRNA sequence [23]. Sense (5*-AAGGAGGTAAAAGAGGAGG-3*) and antisense (5*-ATGTCATAGTGTCCTGCTCTGG-3*) primers were used to amplify this portion of the RARg mRNA, which encodes parts of the hinge and ligand binding regions. This 358-bp region is conserved between the seven reported mouse RARg mRNA isoforms. In experiments detecting RARa and RARg mRNA, the identity of PCR products of the predicted size was verified by automated DNA sequencing performed by the Recombinant DNA Laboratory Core Facility of the Sealy Center for Molecular Science, UTMB. Northern blot analysis. Northern blot analysis was used to detect the presence of mRNA for the various RARs in total RNA extracted from untreated MC-26 and LoVo cells. In these experiments, cells grown in 10-cm culture dishes were initially washed twice with phosphate-buffered saline. Total RNA was then prepared from the untreated cells using guanidinium thiocyanate-phenol-chloroform extraction as described by Chomczynski [24]. The total RNA samples were electrophoresed on 1% agarose, 0.66 M formaldehyde gels. The RNA present in the gel was blotted onto a nylon membrane (MSI, Magnagraph) by capillary transfer and immobilized on the membrane with a UVC-508 ultraviolet crosslinker (Ultra-Lum, Carson, CA). The membrane was next incubated in prehybridization buffer (61 SSPE, 51 Denhardts (0.01% Ficoll, 0.01% polyvinylpyrrolidone, 0.01% bovine serum albumin), 0.1% SDS, and 100 mg/ml salmon sperm DNA) for 3 h at 657C and exposed to a [32P]dCTP-labeled cDNA probe in the same buffer for 12–16 h at 657C. Following this incubation, the membrane was washed under high stringency conditions at 657C (21 SSC for 15 min; 21 SSC, 0.1% SDS for 15 min; 0.21 SSC, 0.1% SDS for 5–10 min) and exposed to Kodak XAR film at 0707C. The probes used to detect the presence of RARa and RARg mRNA were [32P]dCTP-labeled, single-stranded cDNA synthesized by PCR in the presence of only a downstream primer (‘‘asymmetric PCR’’) [25]. The RARa probe was complementary to the region specified by base pairs 1194–1500 in the published RARa sequence [19]. For the RARg probe, a cDNA was synthesized that would bind to the region specified by base pairs 820–1177 in the RARg published sequence [23]. RT-PCR fragments generated from mouse colon cancer cells were used as the templates in the RARa and RARg labeling reactions. Statistics. Statistical significance was determined using one-way analysis of variance with post-hoc Bonferroni t test. A P value õ 0.05 was considered statistically significant.
RESULTS
Regulation of colon cancer cell proliferation by retinoids. To test the ability of the retinoid, atRA, to alter the proliferation of MC-26 and LoVo colon cancer cell lines, each cell line was exposed to a range of
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FIG. 1. atRA stimulates the proliferation of MC-26 cells but does not modulate LoVo cell proliferation. MC-26 (A) and LoVo (B) cells were treated with each concentration of atRA over a 4-day period. During this time period, atRA produced a dose-dependent increase in MC-26 cell growth. The growth of LoVo cells was not significantly altered by atRA. Each bar represents the mean { SEM for 3–4 wells. For each cell line, similar results have been obtained in two other experiments. *P õ 0.05 compared to Control. The amount of DNA in the control samples equalled 1.406 { 0.149 and 1.143 { 0.115 mg/well for the MC-26 and LoVo cells, respectively.
concentrations (10010 – 1007 M) of atRA for 4 days. Unlike breast cancer cells and other tumor cell types that are growth-inhibited by retinoids [3 – 5, 26], the MC26 cells were stimulated to proliferate in the presence of atRA (Fig. 1A). This increase in growth occurred in a dose-dependent manner, with the maximum effect (Ç50% above control) being produced by a concentration of 1008 M atRA. The presence of 10010 –1007 M atRA did not significantly alter the growth of the LoVo cell line within the 4-day treatment period (Fig. 1B). At each concentration tested, the level of DNA/well in LoVo cells exposed to atRA was comparable to that of control cells. The ability of another retinoid, 9-cis-retinoic acid (9cis-RA), to alter the growth of the two cells lines was examined to determine if the growth stimulation produced by atRA could be elicited by other retinoids. The proliferative effects produced by 9-cis-RA were similar to those generated by atRA in the two colon cancer cell lines. Within a 4-day period, 9-cis-RA did not modulate the growth of the LoVo cell line over the concentration range tested (10010 –1007 M) (Fig. 2B). However, analogous to atRA, 9-cis-RA did increase the proliferation of
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the MC-26 cells (Fig. 2A). Compared to atRA, 9-cis-RA was slightly more effective in stimulating MC-26 cell growth (Ç160% of control). However, the maximal effect elicited by both compounds was produced by a concentration of 1008 M retinoid, indicating that the two compounds are equipotent in increasing the proliferation of the MC-26 cell line. Specificity of retinoid-induced growth stimulation. To determine if the increase in MC-26 cell proliferation produced by retinoids also occurred with ligands for other steroid hormone receptors, we examined the effects of the steroids hydrocortisone and progesterone as well as the sterol 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on MC-26 cell growth. Progesterone at a concentration of 1008 M did not significantly alter MC-26 cell proliferation within the 4-day treatment period (Fig. 3). Hydrocortisone and 1,25(OH)2D3 did modulate the proliferation of MC-26 cells; both compounds at a concentration of 1008 M inhibited the growth of MC-26 cells (Fig. 3). Therefore, the stimulation of MC-26 cell proliferation by ligands of the steroid hormone receptors appears to be ligand/steroid-specific.
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FIG. 2. 9-cis-RA enhances the proliferation of MC-26 cells but does not alter the growth of LoVo cells. MC-26 (A) and LoVo (B) cells were treated with each concentration of 9-cis-RA over a 4-day period. 9-cis-RA produced a dose-dependent increase in MC-26 cell growth within this time period. No significant alteration of LoVo cell proliferation was noted after 4 days of 9-cis-RA exposure. For (A), each bar represents the mean { SEM of 7–8 wells. For (B), each bar represents the mean { SEM of 3–4 wells; similar results have been obtained in two other experiments. *P õ 0.05 compared to Control. The amount of DNA in the control samples equalled 1.559 { 0.303 and 0.848 { 0.175 mg/well for the MC-26 and LoVo cells, respectively.
Characterization of retinoic acid receptors involved in retinoid-induced increases in colon cancer cell growth. The growth-stimulatory effects we found in the mouse MC-26 cell line differed not only from the growth-inhibitory effects produced by retinoids in several other cancer cell types but also from the lack of a growth response seen in the LoVo cells. We hypothesized that the different growth responses seen between these cancer cells might be due to differential expression of retinoid receptor subtypes. In an attempt to understand the mechanism underlying retinoid-induced increases in MC-26 cell growth, we began to characterize the retinoid receptors present in both the MC-26 and LoVo cell lines. atRA and 9-cis-RA were equipotent in stimulating MC-26 cell growth. Because these two retinoids have been shown to activate the RARs with equal potency, we examined the RAR subtypes which were present within the two cell lines. RT-PCR was used as an initial screen for the presence of mRNA for the different RAR subtypes (a, b, and g) in the two cell lines. For the RARa, the primers selected were designed to amplify part of the ligand binding region unique to the RARa and common to
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all RARa mRNA isoforms. These primers produced an amplimer of the predicted size, 306 bp, from total RNA isolated from MC-26 cells, indicating that one or more RARa mRNA isoforms are expressed in this cell line (Fig. 4A). Because the primers chosen for amplification of RARa in the mouse cell line are greater than 95% homologous to the corresponding sequence found in human RARa mRNA, they were also utilized to detect the presence of RARa mRNA in the LoVo cell line. Similar to the MC-26 cells, a 306-bp amplimer was obtained in RT-PCR experiments using total RNA extracted from LoVo cells (Fig. 4A). In addition, Northern blot analysis revealed bands similar in size (3.6 and 2.6 kb) to those previously reported for RARa mRNA in total RNA samples prepared from both LoVo and MC-26 cells (Fig. 4B). Thus, these experiments demonstrate the presence of RARa mRNA in both the retinoid-unresponsive LoVo cells and the MC-26 cells, which are growth-stimulated by retinoids. The detection of a substantial amount of RARa mRNA within the MC-26 cell line suggested that one or more of the RARas may be involved in the growth
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FIG. 3. Progesterone (Pg), hydrocortisone (HC), and 1,25(OH)2D3 (VD) do not stimulate the proliferation of MC-26 cells. MC-26 cells were exposed to either 1008 M progesterone (Pg), 1008 M hydrocortisone (HC), or 1008 M 1,25(OH)2D3 (VD) for 4 days. Within this time period, the progesterone effect to decrease growth was not statistically significant. Hydrocortisone and 1,25(OH)2D3 produced significant decreases in MC-26 cell proliferation. Each bar represents the mean { SEM for 4–5 wells. *P õ 0.05 compared to Control. The amount of DNA for the control samples equalled 0.144 { 0.029 mg/ well.
stimulation produced by retinoids in MC-26 cells. In breast cancer cells, the RARa is thought to be one of the RARs which mediates growth inhibitory effects pro-
FIG. 4. (A) RT-PCR detection of RARa mRNA. The predicted amplimer size is 306 bp. Lane A, 123 bp marker; lane B, MC-26 cells; lane D, LoVo cells. Lanes C and E contain PCR products in which the amplification protocol was performed in the absence of AMV reverse transcriptase. These lanes served as a negative control to verify that the PCR products could not have been obtained through amplification of genomic RARa sequence. (B) Northern blot detection of RARa mRNA. The cDNA probe was able to detect two mRNA species, 2.6 and 3.6 kb, in total RNA samples prepared from both MC-26 and LoVo cells. Twenty micrograms of total RNA were loaded in each lane of the gel.
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FIG. 5. The selective RARa antagonist Ro 41-5253 does not block the atRA-induced increase in MC-26 cell growth. MC-26 cells were exposed to 1008 M atRA in the presence or absence of each concentration of Ro 41-5253 for 4 days. During this time period, neither concentration of Ro 41-5253 was able to prevent the stimulation of cell proliferation produced by 1008 M atRA. Each bar represents the mean { SEM for four wells. A similar lack of inhibition has been obtained in two other experiments. *P õ 0.05 compared to control (atRA Å 0, Ro 41-5253Å 0). The amount of DNA in the control sample equalled 7.502 { 0.123 g/well.
duced by atRA [27, 28]. To determine whether RARa might be involved in retinoid regulation of MC-26 cell proliferation, the cells were treated for 4 days with 1008 M atRA in the presence of either dimethylsulfoxide vehicle or an RARa-selective antagonist, Ro 41-5253. Within this time period, neither concentration of Ro 41-5253 tested (1007 and 1006 M) was able to block the increase in MC-26 cell proliferation induced by atRA (Fig. 5). These same concentrations were able to suppress biological effects produced by 1008 M atRA in at least two other cell systems [29, 30]. Our results suggest that the RARa does not mediate the growth stimulatory effects of retinoids in MC-26 cells. The primers chosen for RT-PCR detection of RARb mRNA in MC-26 cells were designed to amplify a 430bp section of RARb mRNA encoding parts of the RARb hinge and ligand binding regions. This set of primers was able to synthesize an amplimer of the correct size from the sample used as a positive control, mouse kidney total RNA (Fig. 6A). Although these primers were very effective in amplifying RARb mRNA from mouse
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from LoVo cells. Taken together, the data from the RTPCR and Northern blot analysis reveal that MC-26 and LoVo cells under basal conditions express mRNA for one or more of the RARgs. Western blot analysis on protein extracts prepared from MC-26 cells demonstrated the presence of proteins of approximately 50 kDa in size that were detected by antibodies to two of the RARg isoforms, the RARg1 and RARg2 (data not shown). This indicates that proteins can be transcribed from the RARg mRNA present in the cell. FIG. 6. RT-PCR detection of RARb mRNA. (A) MC-26 cells contain very little, if any, RARb mRNA. The predicted size of the amplimer is 430 bp. Lane A, 100-bp marker; lane B, MC-26 cells; lane C, mouse kidney. Mouse kidney total RNA samples were used as a positive control. (B) RARb mRNA is expressed in LoVo cells. The predicted amplimer size is 253 bp. Lane A, 100-bp marker; lane B, LoVo cells; lane C, MCF-7 cells. Total RNA from the MCF-7 breast cancer cell line was used as a negative control in these experiments.
kidney, they were only able to produce a very faint 430bp band when total RNA extracted from MC-26 cells was used as the initial substrate (Fig. 6A). This suggests that very little, if any, RARb mRNA is expressed in the MC-26 cell line under basal conditions. The primers used to detect the RARb in MC-26 cells were not very homologous to the reported human RARb cDNA sequence; as a result, a second set of primers was chosen to detect the presence of RARb mRNA in the LoVo cell line. With these primers, we were able to obtain an amplimer of the predicted size, 253 bp, as well as other nonspecific bands in total RNA samples prepared from LoVo cells (Fig. 6B). The presence of a correctly sized PCR product suggests that LoVo cells express one or more isoforms of RARb mRNA. To detect RARg mRNA, two oligonucleotide primers were selected that would amplify a section of RARg mRNA that encodes part of the hinge and ligand binding regions of these receptors. These primers produced an amplimer of the correct size, 358 bp, in total RNA samples prepared from both the positive control, mouse kidney, and MC-26 cells (Fig. 7A). RARg mRNA could also be detected on Northern blots containing untreated MC-26 cells (Fig. 7B). Two mRNA species, approximately of size 3.1 and 1.6 kb, were detected with our RARg cDNA probe. The 3.1-kb species has been previously detected in other cell systems [31, 32]. The low homology between the mouse and human RARg mRNAs in the region encoding the PCR primers precluded the use of these primers for the detection of RARg in the LoVo cell line. However, because the region between the primers was relatively homologous, the cDNA probe used for Northern blot analyses in MC26 cells was able to detect the presence of RARg mRNA in total RNA samples prepared from LoVo cells (Fig. 7B). mRNA species of 3.1 and 1.6 kb were also detected with this cDNA probe in total RNA samples prepared
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DISCUSSION
In several tissues and cell lines, retinoids at high pharmacological concentrations (1007 –1006 M) have been shown to inhibit cell proliferation and, in some cases, induce apoptosis [3–5, 26]. The ability of these compounds to block the growth of cultured cancer cells has led to the concept that retinoids may be useful as cancer chemotherapeutic agents. The data presented here, however, suggest that some colon cancer cells may not respond to retinoids with an inhibition of cell growth. In our study, both atRA and 9-cis-RA increased the proliferation of the MC-26 colon cancer cell line. This growth stimulation was dose-dependent and peaked at 1008 M, a physiological concentration for retinoids [33]. The growth-stimulatory effects of retinoids noted in our experiments were obtained in the mouse MC-26 colon cancer cell line. The MC-26 cell line is derived from a 1,2-dimethylhydrazine-induced mouse colon adenocarcinoma [16]. Spjut has demonstrated that colon tumors induced by chemicals such as 1,2-dimethylhydrazine are histologically similar to the adenocarcinomas that form in the human colon [34]. The distribu-
FIG. 7. (A) RT-PCR detection of RARg mRNA. The predicted size of the amplimer is 358 bp. Lane A, 100-bp marker; lane B, MC26 cells; lane C, mouse kidney. Samples obtained from mouse kidney served as a positive control in these experiments. (B). Northern blot detection of RARg mRNA. The cDNA probe was able to detect two mRNA species, 3.1 and 1.6 kb, in total RNA samples prepared from both MC-26 (A) and LoVo (B) cells. Twenty micrograms of total RNA were loaded in each lane of the gel.
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tion of these tumors along the length of the mouse colon is also comparable to the location of colon tumors found in the human, where tumors are commonly found in both the proximal and distal regions of the colon [34]. Our results demonstrate that the MC-26 cell line is also similar to human colon cancer cells in its response to retinoids, because there have been two other reports which have described growth-stimulatory effects produced by retinoids in cultured human colon cancer cells. Work done by Kane et al. [35] has demonstrated that the incorporation of [3H]thymidine, another measure of cell proliferation, is increased in the human HT29 colon cancer cell line by 1009 –1007 M 9-cis-RA. In addition, the growth of the SW 948 human colon cancer cell line, as measured by cell number, is enhanced by micromolar concentrations of atRA and other synthetic retinoids [8]. The colon appears not to be the only tumor cell type whose growth can be stimulated by retinoids. While enhanced proliferation in the presence of retinoids is a rare occurrence, physiological concentrations (1008 M) of atRA enhance the growth of the prostatic cancer cell line LNCaP [2, 36]. The proliferation of GH-1 pituitary tumor cells [37] is also increased when these cells are exposed to atRA. This suggests that while retinoids may suppress the progression of some cancers, they could promote the growth of malignant cells found in the colon and other tissues. The RARs and RXRs are believed to mediate the majority of biological effects produced by retinoids. In addition to forming homodimers, RARs and RXRs can bind together to form heterodimers which can activate gene transcription [38–42]. Both atRA and 9-cis-RA can bind to RARs; the RXRs, however, have only been shown to bind 9-cis-RA. In transactivation assays measuring receptor function, 9-cis-RA acts as a full agonist and is equipotent in activating both RARs and RXRs. atRA is an ineffective agonist for RXRs in these assays; however, it acts as an agonist with an efficacy and potency comparable to 9-cis-RA for RARs [43]. If RXR homodimers were the only type of retinoid receptor complex involved in the proliferative effects of retinoids in MC-26 cells, one would expect 9-cis-RA, the naturally occurring RXR ligand, to be a more potent activator of cell growth than atRA. However, our data demonstrate that atRA and 9-cis-RA are equipotent in increasing MC-26 cell proliferation. Thus, the responses we have measured most likely involve activation of a RAR. Since DNA binding and gene activation by RARs is greatly enhanced and, in some cases, requires heterodimerization with an RXR [39, 41, 44, 45], our data do not eliminate the possibility that RXRs play a role in the growth stimulatory effects elicited by retinoids. Our data demonstrate that there are significant levels of RARa and RARg mRNA present in MC-26 cells, while RARb mRNA is expressed at very low levels. The
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very low to nonexistent basal expression of the RARb mRNA in the MC-26 cells has led us to hypothesize that the RARb may not play a significant role in retinoidinduced increases in MC-26 colon cancer cell growth. This hypothesis is supported by recent data which suggest the RARb is the direct mediator of the growth inhibitory effect produced by retinoids in breast cancer cells [4]. It may be that in cells where the RARb functions to regulate proliferative responses to retinoids, the net effect is growth inhibition. Like the RARb, the RARa has been associated with the growth inhibitory effects of retinoids in breast cancer cells [27, 28]. While the RARb plays a direct role in the growth inhibitory effect of retinoids, the RARa has been shown to modulate this response via regulation of RARb mRNA and protein levels [4]. To test whether the RARa is involved in the retinoid-induced increases in colon cancer cell growth, we examined the ability of an RARa-selective antagonist to inhibit the growth effect. This RARa antagonist, Ro 41-5253, has been shown in transactivation assays to selectively block biological effects mediated by the RARa, while having little effect on the RARb and RARg [29]. However, in MC-26 cells, Ro 41-5253 was unable to prevent the atRA-induced growth effect. These data suggest that the RARa is not involved in retinoid-induced increases in MC-26 colon cancer cell growth. It is possible that the RARg may mediate the growth effects of retinoids in the MC-26 cell line. We have been able to detect both mRNA and protein for the RARg within MC-26 cells. The lack of selective antagonists for the RARg, however, has prevented the further characterization of the involvement of RARg in the retinoidinduced increase in MC-26 cell proliferation at this time. Future experiments involving antisense oligonucleotides designed to deplete RARg levels may be useful in addressing the role of the RARg in retinoid-mediated increases in MC-26 cell growth. Although retinoids increased the proliferation of the MC-26 cell line, the growth of the LoVo cell line was not significantly modulated in the presence of retinoids. Therefore, our data demonstrate that growth stimulation is not produced by retinoids in every colon cancer cell line. Work done by Kane et al. [35] also showed that colon cancer cells can vary in their response to retinoids. In their studies, the proliferative rate of the human HT-29 colon cancer cell line was enhanced in the presence of 9-cis-RA. Caco-2 human colon cancer cells, however, responded to 9-cis-RA with a decrease in proliferation, as measured by [3H]thymidine incorporation. Additional data supporting the growth inhibitory effects of retinoids in Caco-2 cells have been obtained by McCormack et al., who have noted growth inhibition by 1008 M atRA after 3 days of retinoid exposure [46]. These studies provide further evidence that
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retinoids do not regulate the growth of all colon cancer cells in the same manner. The inability of the LoVo cells to respond to retinoids does not appear to be due to the absence of RARs within this cell line. LoVo cells possess the ability to synthesize the same retinoic acid receptor subtypes that are present in the MC-26 cell line. While both cell lines express some RARa, RARb, and RARg mRNA, there may be isoforms of these RAR subtypes present in MC26 cells which are absent in the LoVo cell line. Differences in RAR isoform expression could therefore explain the difference in retinoid responsiveness between the two cell lines. Another possibility is that the ability of retinoid receptors to interact with other signaling pathways could differ between the two colon cancer cell lines. Evidence supporting this hypothesis has been obtained in experiments performed by Rosewicz et al. [47] in human pancreatic carcinoma cells. This group was unable to detect retinoid receptor subtype differences between AsPc1 and Capan 2 cells, two pancreatic carcinoma cell lines which respond differently to atRA. They did, however, notice that in the AsPc1 cells, atRA induced expression of protein kinase Ca (PKCa), a protein kinase C subtype they have shown to be involved in the growth stimulation produced by atRA within this cell line [47]. Conversely, PKCa protein expression was decreased by atRA in the Capan 2 cells, whose growth is inhibited by atRA. The data collected by these authors therefore suggest that the net growth effect produced by retinoids in pancreatic carcinomas may be determined by the cell’s ability to modulate protein kinase C expression. In a likewise manner, retinoids in colon cancer cells may modulate the expression of protein kinases or other proteins which are required for retinoid-induced increases in cell growth. The growth response elicited by retinoids in colon cancer cells may therefore depend not only upon the retinoid receptor subtypes but also on what accessory proteins are expressed in the presence of retinoids. In summary, our data demonstrate that colon cancer cells differ in their ability to respond to both atRA and 9-cis-RA. The growth of LoVo cells are unaffected by the presence of retinoids, while the growth of MC-26 cells is enhanced following retinoid exposure. This variability suggests that retinoids may not be useful as therapeutic agents for colon cancer. Furthermore, care must be exercised when designing retinoid therapies for other types of cancers so that the growth of tumors that might be present in the colon will not be stimulated. We thank Drs. M. Klaus (Basel, Switzerland) and Jerry Sepinwall (Nutley, New Jersey) of Hoffman–LaRoche for their generous gift of the RARa antagonist Ro 41-5253 and 9-cis-retinoic acid, respectively.
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38. Zhang, X.-k., Hoffmann, B., Tran, P., Graupner, G., and Pfahl, M. (1992) Nature 355, 441–446. 39. Marks, M. S., Hallenbeck, P. L., Nagata, T., et al. (1992) EMBO J. 11, 1419–1435. 40. Kliewer, S. A., Umesono, K., Mangelsdorf, D. J., and Evans, R. M. (1992) Nature 355, 446–449. 41. Bugge, T. H., Pohl, J., Lonnoy, O., and Stunnenberg, H. G. (1992) EMBO J. 11, 1409–1418. 42. Yu, V. C., Delsert, C., Andersen, B., et al. (1991) Cell 67, 1251– 1266. 43. Allenby, G., Bocquel, M.-T., Saunders, M., et al. (1993) Proc. Natl. Acad. Sci. USA 90, 30–34. 44. Leid, M., Kastner, P., Lyons, R., et al. (1992) Cell 68, 377–395. 45. Hermann, T., Hoffmann, B., Zhang, X.-K., Tran, P., and Pfahl, M. (1992) Mol. Endocrinol. 6, 1153–1162. 46. McCormack, S. A., Viar, M. J., Tague, L., and Johnson, L. R. (1996) In Vitro Cell. Dev. Biol.-Animal 32, 53–61. 47. Rosewicz, S., Brembeck, F., Kaiser, A., Marschall, Z. V., and Riecken, E.-O. (1996) Endocrinology 137, 3340–3347.
Received September 6, 1996 Revised version received January 13, 1997
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Retinoids Differentially Regulate the Proliferation of Colon Cancer Cell Lines LaMonica V. Stewart and Mary L. Thomas1 Department of Pharmacology and Toxicology, Route J-31, University of Texas Medical Branch, Galveston, Texas 77555-1031
In this study, the proliferative effects of retinoids were examined in the MC-26 and LoVo colon adenocarcinoma cell lines. The proliferation of the LoVo cell line was not altered in the presence of the retinoids all trans-retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA). Both retinoids, however, stimulated the growth, as measured by cell proliferation, of MC-26 cells. atRA and 9-cis-RA were equipotent in increasing MC-26 cell proliferation, suggesting that the growth stimulation is mediated by one or more retinoic acid receptors (RARs). To determine the RAR which might be responsible for this growth stimulatory effect, we characterized the RAR subtypes which were present in both cell lines. mRNA for the RARa, RARb, and RARg were detected in the MC-26 cell. Of the RARs present in MC-26 cells, the RARa does not mediate the growth stimulatory effects of retinoids, for a selective RARa antagonist was unable to prevent the retinoidinduced increase in MC-26 cell growth. RARa, RARb, and RARg mRNA are also expressed in the LoVo cell line; the lack of growth-stimulation by retinoids in LoVo cells, therefore, does not seem to be due to the absence of RARs. The results obtained in these experiments demonstrate that the growth response elicited by retinoids can vary between colon cancer cells and that the differences in response may not be solely determined by the RAR subtypes which are expressed in a colon cancer cell line. q 1997 Academic Press
INTRODUCTION
One class of compounds which has been shown to play a key role in the regulation of cell growth is the retinoids. Retinoids are metabolites of vitamin A, a nutrient derived from carotenoids and retinyl esters present in the diet [1]. The biologically active retinoids, which include all trans-retinoic acid (atRA) and 9-cisretinoic acid (9-cis-RA), are believed to alter cell function via interactions with two types of receptors belonging to the steroid hormone receptor superfamily: the 1 To whom correspondence and reprint requests should be addressed. Fax: (409) 772-9642. E-mail: [email protected].
retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). In vitro experiments have demonstrated that retinoids inhibit the proliferation of several types of tumor cells grown in culture, including cell lines derived from teratocarcinomas, human myelocytic leukemias, breast tumors, and tumors of the prostate [2– 5]. The growth inhibitory effects of retinoids are also evident in vivo. For example, the incidence of chemically induced mammary tumors in rodents was decreased in animals treated with either atRA or 9-cisRA [6, 7]. Because retinoids are successful in preventing tumor development in vivo and tumor cell growth in vitro, analogs of the retinoids are presently being developed for use in humans as an anticancer therapy [3, 8]. The effects of retinoids on the growth and development of colon cancer have not been extensively examined. Conflicting data have been obtained from in vivo experiments studying the effects of retinoids on the development of chemically induced colon tumors in rats [9–12]. There are also few reports that detail the effects of retinoids on colon cancer cells in culture. It is therefore difficult to determine whether the inhibitory growth effects of retinoids produced in other types of tumors will also occur in colon cancer cells and tumors. To gain a better understanding of how retinoids might affect colon cancer cells, we examined the ability of biologically active retinoids to modulate the proliferation of two colon cancer cell lines, MC-26 and LoVo cells. We have previously demonstrated MC-26 cells can be growth-stimulated by physiological concentrations of the steroid 17-b-estradiol (E2) while LoVo cells are not [13, 14]. The proliferation of LoVo cells can also be modulated by compounds acting via steroid hormone receptors, for the growth of these cells has been shown to be inhibited by the sterol 1,25-dihydroxyvitamin D3 [15]. The results obtained in the present study reveal that different proliferative responses are produced by retinoids in these two colon cancer cell lines. MATERIALS AND METHODS Cell culture. MC-26 cells, a mouse colon adenocarcinoma derived from a 1,2-dimethylhydrazine-induced tumor [16], were obtained
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0014-4827/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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from the Department of Surgery at the University of Texas Medical Branch (UTMB). They were maintained in phenol red-free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% fetal bovine serum (FBS) (Atlanta Biological), 10 U/ml penicillin, and 0.01 mg/ml streptomycin in T-75 culture flasks (Corning). Cell passages between 30 and 40 were used in all experiments. The medium was changed every 2 days. LoVo cells, a human colon adenocarcinoma derived from a metastatic tumor nodule from a 56-year-old Caucasian male, were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were maintained in Hams F-12 medium supplemented with 10% FBS in T-75 culture flasks. Cells between passage 30 and 40 were used in all experiments. The medium was changed every 3 days. All cultured cells were kept at 377C in a 95% air/5% CO2 environment. They were subcultured weekly 1:10. Cell proliferation experiments. LoVo and MC-26 cells were plated at a density of 8,000–10,000 cells/well in 24-well culture plates and allowed to attach for 24 h. The medium was then changed (Day ‘‘0’’) to DMEM supplemented with 10% NuSerum, 10 U/ml penicillin, and 0.01 mg/ml streptomycin, and either retinoids or other steroid hormones dissolved in absolute ethanol were added to the medium. The final ethanol concentration in the wells was less than 0.1%. Control cells received an equal concentration of ethanol vehicle. To prevent degradation of the light-sensitive retinoids, drugs were added to the culture medium under subdued light. The medium was changed and fresh drug was added every 2 days. On Day 4, the cells were washed two times with phosphate-buffered saline and lysed by freeze-thawing the cells two times at 0707C. The lysates were further dispersed by placing them in a sonicating bath for 15 min. The amount of DNA in each well was then measured using the fluorescent dye, Hoechst No. 33258 (Sigma). An excitation wavelength of 356 nm and an emission wavelength of 458 nm was used to measure fluorescence [17]. In experiments involving the RARa antagonist, Ro 41-5253, the cells were pretreated for approximately 2 min with either RARa antagonist or dimethylsulfoxide (DMSO) vehicle. The retinoids or ethanol vehicle were then added to the wells. The final concentration of DMSO in the culture wells did not exceed 0.1%. The antagonist remained in the culture system for the duration of the experiment and was reapplied to the cultures whenever the medium was changed. Reverse transcription-polymerase chain reaction detection of retinoic acid receptor mRNAs. A GeneAmp Thermocycler (model 9600, Perkin–Elmer–Cetus) was employed for the reverse transcription and polymerase chain reactions. A computer algorithm [18] was used to select the sense and antisense oligonucleotide primers used in each experiment. The reverse transcription reaction was conducted in the presence of 0.5 mg total cellular RNA, AMV reverse transcriptase, and the antisense primer (0.75 mM) in a final volume of 10 ml for 30 min at 427C. The reverse transcriptase next was inactivated by heating at 957C for 5 min. PCR reagents, Taq DNA polymerase, and the sense primer (0.15 mM) were then added to a final volume of 50 ml, and 35 cycles of PCR amplification were performed (5 cycles at 947C for 15 s, 657C for 20 s, 727C for 15 s; 30 cycles at 947C for 15 s, 557C for 20 s, 727C for 15 s). The amplified products were analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide. A 306-bp fragment of the RARa mRNA was selected for amplification based on the published sequence for the mouse RARa cDNA [19]. Sense (5*-CGAACAACAGCTCAGAACAACG-3*) and antisense (5*-AAGGCAAAGACCAAGTCGG-3*) primers were chosen to amplify this fragment, which encodes a portion of the RARa ligand binding region common to all of the reported RARa isoforms. When compared to the published human RARa sequence, the sense primer is 100% homologous to the human RARa sequence, and there is
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only one base pair mismatch between the antisense primer and the human sequence. From the published mouse RARb2 mRNA sequence [20], both sense (5*-GAGCCTTCAAAGCAGGAATGC-3*) and antisense (5*TGAACACAAGGTCAGTCAGAGG-3*) primers were chosen to amplify a 430-bp region of RARb mRNA. This portion of RARb mRNA includes parts of the hinge and ligand binding regions and is present in all of the reported mouse RARb mRNA isoforms. A second set of sense (5*-GAAGCAAGAATGCACAGAGAGC-3*) and antisense (5*GATGGTCAAGCCAGTGAAACC-3*) primers was used to detect the presence of RARb in the human LoVo cell line. Based on the published human RARb cDNA sequence [21, 22], these primers were designed to amplify a 253-bp region of the hinge and ligand binding domains that is common to all of the reported human RARb isoforms. A 358-bp section of the RARg was chosen for amplification based on the published mouse RARg mRNA sequence [23]. Sense (5*-AAGGAGGTAAAAGAGGAGG-3*) and antisense (5*-ATGTCATAGTGTCCTGCTCTGG-3*) primers were used to amplify this portion of the RARg mRNA, which encodes parts of the hinge and ligand binding regions. This 358-bp region is conserved between the seven reported mouse RARg mRNA isoforms. In experiments detecting RARa and RARg mRNA, the identity of PCR products of the predicted size was verified by automated DNA sequencing performed by the Recombinant DNA Laboratory Core Facility of the Sealy Center for Molecular Science, UTMB. Northern blot analysis. Northern blot analysis was used to detect the presence of mRNA for the various RARs in total RNA extracted from untreated MC-26 and LoVo cells. In these experiments, cells grown in 10-cm culture dishes were initially washed twice with phosphate-buffered saline. Total RNA was then prepared from the untreated cells using guanidinium thiocyanate-phenol-chloroform extraction as described by Chomczynski [24]. The total RNA samples were electrophoresed on 1% agarose, 0.66 M formaldehyde gels. The RNA present in the gel was blotted onto a nylon membrane (MSI, Magnagraph) by capillary transfer and immobilized on the membrane with a UVC-508 ultraviolet crosslinker (Ultra-Lum, Carson, CA). The membrane was next incubated in prehybridization buffer (61 SSPE, 51 Denhardts (0.01% Ficoll, 0.01% polyvinylpyrrolidone, 0.01% bovine serum albumin), 0.1% SDS, and 100 mg/ml salmon sperm DNA) for 3 h at 657C and exposed to a [32P]dCTP-labeled cDNA probe in the same buffer for 12–16 h at 657C. Following this incubation, the membrane was washed under high stringency conditions at 657C (21 SSC for 15 min; 21 SSC, 0.1% SDS for 15 min; 0.21 SSC, 0.1% SDS for 5–10 min) and exposed to Kodak XAR film at 0707C. The probes used to detect the presence of RARa and RARg mRNA were [32P]dCTP-labeled, single-stranded cDNA synthesized by PCR in the presence of only a downstream primer (‘‘asymmetric PCR’’) [25]. The RARa probe was complementary to the region specified by base pairs 1194–1500 in the published RARa sequence [19]. For the RARg probe, a cDNA was synthesized that would bind to the region specified by base pairs 820–1177 in the RARg published sequence [23]. RT-PCR fragments generated from mouse colon cancer cells were used as the templates in the RARa and RARg labeling reactions. Statistics. Statistical significance was determined using one-way analysis of variance with post-hoc Bonferroni t test. A P value õ 0.05 was considered statistically significant.
RESULTS
Regulation of colon cancer cell proliferation by retinoids. To test the ability of the retinoid, atRA, to alter the proliferation of MC-26 and LoVo colon cancer cell lines, each cell line was exposed to a range of
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FIG. 1. atRA stimulates the proliferation of MC-26 cells but does not modulate LoVo cell proliferation. MC-26 (A) and LoVo (B) cells were treated with each concentration of atRA over a 4-day period. During this time period, atRA produced a dose-dependent increase in MC-26 cell growth. The growth of LoVo cells was not significantly altered by atRA. Each bar represents the mean { SEM for 3–4 wells. For each cell line, similar results have been obtained in two other experiments. *P õ 0.05 compared to Control. The amount of DNA in the control samples equalled 1.406 { 0.149 and 1.143 { 0.115 mg/well for the MC-26 and LoVo cells, respectively.
concentrations (10010 – 1007 M) of atRA for 4 days. Unlike breast cancer cells and other tumor cell types that are growth-inhibited by retinoids [3 – 5, 26], the MC26 cells were stimulated to proliferate in the presence of atRA (Fig. 1A). This increase in growth occurred in a dose-dependent manner, with the maximum effect (Ç50% above control) being produced by a concentration of 1008 M atRA. The presence of 10010 –1007 M atRA did not significantly alter the growth of the LoVo cell line within the 4-day treatment period (Fig. 1B). At each concentration tested, the level of DNA/well in LoVo cells exposed to atRA was comparable to that of control cells. The ability of another retinoid, 9-cis-retinoic acid (9cis-RA), to alter the growth of the two cells lines was examined to determine if the growth stimulation produced by atRA could be elicited by other retinoids. The proliferative effects produced by 9-cis-RA were similar to those generated by atRA in the two colon cancer cell lines. Within a 4-day period, 9-cis-RA did not modulate the growth of the LoVo cell line over the concentration range tested (10010 –1007 M) (Fig. 2B). However, analogous to atRA, 9-cis-RA did increase the proliferation of
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the MC-26 cells (Fig. 2A). Compared to atRA, 9-cis-RA was slightly more effective in stimulating MC-26 cell growth (Ç160% of control). However, the maximal effect elicited by both compounds was produced by a concentration of 1008 M retinoid, indicating that the two compounds are equipotent in increasing the proliferation of the MC-26 cell line. Specificity of retinoid-induced growth stimulation. To determine if the increase in MC-26 cell proliferation produced by retinoids also occurred with ligands for other steroid hormone receptors, we examined the effects of the steroids hydrocortisone and progesterone as well as the sterol 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on MC-26 cell growth. Progesterone at a concentration of 1008 M did not significantly alter MC-26 cell proliferation within the 4-day treatment period (Fig. 3). Hydrocortisone and 1,25(OH)2D3 did modulate the proliferation of MC-26 cells; both compounds at a concentration of 1008 M inhibited the growth of MC-26 cells (Fig. 3). Therefore, the stimulation of MC-26 cell proliferation by ligands of the steroid hormone receptors appears to be ligand/steroid-specific.
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FIG. 2. 9-cis-RA enhances the proliferation of MC-26 cells but does not alter the growth of LoVo cells. MC-26 (A) and LoVo (B) cells were treated with each concentration of 9-cis-RA over a 4-day period. 9-cis-RA produced a dose-dependent increase in MC-26 cell growth within this time period. No significant alteration of LoVo cell proliferation was noted after 4 days of 9-cis-RA exposure. For (A), each bar represents the mean { SEM of 7–8 wells. For (B), each bar represents the mean { SEM of 3–4 wells; similar results have been obtained in two other experiments. *P õ 0.05 compared to Control. The amount of DNA in the control samples equalled 1.559 { 0.303 and 0.848 { 0.175 mg/well for the MC-26 and LoVo cells, respectively.
Characterization of retinoic acid receptors involved in retinoid-induced increases in colon cancer cell growth. The growth-stimulatory effects we found in the mouse MC-26 cell line differed not only from the growth-inhibitory effects produced by retinoids in several other cancer cell types but also from the lack of a growth response seen in the LoVo cells. We hypothesized that the different growth responses seen between these cancer cells might be due to differential expression of retinoid receptor subtypes. In an attempt to understand the mechanism underlying retinoid-induced increases in MC-26 cell growth, we began to characterize the retinoid receptors present in both the MC-26 and LoVo cell lines. atRA and 9-cis-RA were equipotent in stimulating MC-26 cell growth. Because these two retinoids have been shown to activate the RARs with equal potency, we examined the RAR subtypes which were present within the two cell lines. RT-PCR was used as an initial screen for the presence of mRNA for the different RAR subtypes (a, b, and g) in the two cell lines. For the RARa, the primers selected were designed to amplify part of the ligand binding region unique to the RARa and common to
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all RARa mRNA isoforms. These primers produced an amplimer of the predicted size, 306 bp, from total RNA isolated from MC-26 cells, indicating that one or more RARa mRNA isoforms are expressed in this cell line (Fig. 4A). Because the primers chosen for amplification of RARa in the mouse cell line are greater than 95% homologous to the corresponding sequence found in human RARa mRNA, they were also utilized to detect the presence of RARa mRNA in the LoVo cell line. Similar to the MC-26 cells, a 306-bp amplimer was obtained in RT-PCR experiments using total RNA extracted from LoVo cells (Fig. 4A). In addition, Northern blot analysis revealed bands similar in size (3.6 and 2.6 kb) to those previously reported for RARa mRNA in total RNA samples prepared from both LoVo and MC-26 cells (Fig. 4B). Thus, these experiments demonstrate the presence of RARa mRNA in both the retinoid-unresponsive LoVo cells and the MC-26 cells, which are growth-stimulated by retinoids. The detection of a substantial amount of RARa mRNA within the MC-26 cell line suggested that one or more of the RARas may be involved in the growth
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FIG. 3. Progesterone (Pg), hydrocortisone (HC), and 1,25(OH)2D3 (VD) do not stimulate the proliferation of MC-26 cells. MC-26 cells were exposed to either 1008 M progesterone (Pg), 1008 M hydrocortisone (HC), or 1008 M 1,25(OH)2D3 (VD) for 4 days. Within this time period, the progesterone effect to decrease growth was not statistically significant. Hydrocortisone and 1,25(OH)2D3 produced significant decreases in MC-26 cell proliferation. Each bar represents the mean { SEM for 4–5 wells. *P õ 0.05 compared to Control. The amount of DNA for the control samples equalled 0.144 { 0.029 mg/ well.
stimulation produced by retinoids in MC-26 cells. In breast cancer cells, the RARa is thought to be one of the RARs which mediates growth inhibitory effects pro-
FIG. 4. (A) RT-PCR detection of RARa mRNA. The predicted amplimer size is 306 bp. Lane A, 123 bp marker; lane B, MC-26 cells; lane D, LoVo cells. Lanes C and E contain PCR products in which the amplification protocol was performed in the absence of AMV reverse transcriptase. These lanes served as a negative control to verify that the PCR products could not have been obtained through amplification of genomic RARa sequence. (B) Northern blot detection of RARa mRNA. The cDNA probe was able to detect two mRNA species, 2.6 and 3.6 kb, in total RNA samples prepared from both MC-26 and LoVo cells. Twenty micrograms of total RNA were loaded in each lane of the gel.
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FIG. 5. The selective RARa antagonist Ro 41-5253 does not block the atRA-induced increase in MC-26 cell growth. MC-26 cells were exposed to 1008 M atRA in the presence or absence of each concentration of Ro 41-5253 for 4 days. During this time period, neither concentration of Ro 41-5253 was able to prevent the stimulation of cell proliferation produced by 1008 M atRA. Each bar represents the mean { SEM for four wells. A similar lack of inhibition has been obtained in two other experiments. *P õ 0.05 compared to control (atRA Å 0, Ro 41-5253Å 0). The amount of DNA in the control sample equalled 7.502 { 0.123 g/well.
duced by atRA [27, 28]. To determine whether RARa might be involved in retinoid regulation of MC-26 cell proliferation, the cells were treated for 4 days with 1008 M atRA in the presence of either dimethylsulfoxide vehicle or an RARa-selective antagonist, Ro 41-5253. Within this time period, neither concentration of Ro 41-5253 tested (1007 and 1006 M) was able to block the increase in MC-26 cell proliferation induced by atRA (Fig. 5). These same concentrations were able to suppress biological effects produced by 1008 M atRA in at least two other cell systems [29, 30]. Our results suggest that the RARa does not mediate the growth stimulatory effects of retinoids in MC-26 cells. The primers chosen for RT-PCR detection of RARb mRNA in MC-26 cells were designed to amplify a 430bp section of RARb mRNA encoding parts of the RARb hinge and ligand binding regions. This set of primers was able to synthesize an amplimer of the correct size from the sample used as a positive control, mouse kidney total RNA (Fig. 6A). Although these primers were very effective in amplifying RARb mRNA from mouse
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from LoVo cells. Taken together, the data from the RTPCR and Northern blot analysis reveal that MC-26 and LoVo cells under basal conditions express mRNA for one or more of the RARgs. Western blot analysis on protein extracts prepared from MC-26 cells demonstrated the presence of proteins of approximately 50 kDa in size that were detected by antibodies to two of the RARg isoforms, the RARg1 and RARg2 (data not shown). This indicates that proteins can be transcribed from the RARg mRNA present in the cell. FIG. 6. RT-PCR detection of RARb mRNA. (A) MC-26 cells contain very little, if any, RARb mRNA. The predicted size of the amplimer is 430 bp. Lane A, 100-bp marker; lane B, MC-26 cells; lane C, mouse kidney. Mouse kidney total RNA samples were used as a positive control. (B) RARb mRNA is expressed in LoVo cells. The predicted amplimer size is 253 bp. Lane A, 100-bp marker; lane B, LoVo cells; lane C, MCF-7 cells. Total RNA from the MCF-7 breast cancer cell line was used as a negative control in these experiments.
kidney, they were only able to produce a very faint 430bp band when total RNA extracted from MC-26 cells was used as the initial substrate (Fig. 6A). This suggests that very little, if any, RARb mRNA is expressed in the MC-26 cell line under basal conditions. The primers used to detect the RARb in MC-26 cells were not very homologous to the reported human RARb cDNA sequence; as a result, a second set of primers was chosen to detect the presence of RARb mRNA in the LoVo cell line. With these primers, we were able to obtain an amplimer of the predicted size, 253 bp, as well as other nonspecific bands in total RNA samples prepared from LoVo cells (Fig. 6B). The presence of a correctly sized PCR product suggests that LoVo cells express one or more isoforms of RARb mRNA. To detect RARg mRNA, two oligonucleotide primers were selected that would amplify a section of RARg mRNA that encodes part of the hinge and ligand binding regions of these receptors. These primers produced an amplimer of the correct size, 358 bp, in total RNA samples prepared from both the positive control, mouse kidney, and MC-26 cells (Fig. 7A). RARg mRNA could also be detected on Northern blots containing untreated MC-26 cells (Fig. 7B). Two mRNA species, approximately of size 3.1 and 1.6 kb, were detected with our RARg cDNA probe. The 3.1-kb species has been previously detected in other cell systems [31, 32]. The low homology between the mouse and human RARg mRNAs in the region encoding the PCR primers precluded the use of these primers for the detection of RARg in the LoVo cell line. However, because the region between the primers was relatively homologous, the cDNA probe used for Northern blot analyses in MC26 cells was able to detect the presence of RARg mRNA in total RNA samples prepared from LoVo cells (Fig. 7B). mRNA species of 3.1 and 1.6 kb were also detected with this cDNA probe in total RNA samples prepared
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DISCUSSION
In several tissues and cell lines, retinoids at high pharmacological concentrations (1007 –1006 M) have been shown to inhibit cell proliferation and, in some cases, induce apoptosis [3–5, 26]. The ability of these compounds to block the growth of cultured cancer cells has led to the concept that retinoids may be useful as cancer chemotherapeutic agents. The data presented here, however, suggest that some colon cancer cells may not respond to retinoids with an inhibition of cell growth. In our study, both atRA and 9-cis-RA increased the proliferation of the MC-26 colon cancer cell line. This growth stimulation was dose-dependent and peaked at 1008 M, a physiological concentration for retinoids [33]. The growth-stimulatory effects of retinoids noted in our experiments were obtained in the mouse MC-26 colon cancer cell line. The MC-26 cell line is derived from a 1,2-dimethylhydrazine-induced mouse colon adenocarcinoma [16]. Spjut has demonstrated that colon tumors induced by chemicals such as 1,2-dimethylhydrazine are histologically similar to the adenocarcinomas that form in the human colon [34]. The distribu-
FIG. 7. (A) RT-PCR detection of RARg mRNA. The predicted size of the amplimer is 358 bp. Lane A, 100-bp marker; lane B, MC26 cells; lane C, mouse kidney. Samples obtained from mouse kidney served as a positive control in these experiments. (B). Northern blot detection of RARg mRNA. The cDNA probe was able to detect two mRNA species, 3.1 and 1.6 kb, in total RNA samples prepared from both MC-26 (A) and LoVo (B) cells. Twenty micrograms of total RNA were loaded in each lane of the gel.
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tion of these tumors along the length of the mouse colon is also comparable to the location of colon tumors found in the human, where tumors are commonly found in both the proximal and distal regions of the colon [34]. Our results demonstrate that the MC-26 cell line is also similar to human colon cancer cells in its response to retinoids, because there have been two other reports which have described growth-stimulatory effects produced by retinoids in cultured human colon cancer cells. Work done by Kane et al. [35] has demonstrated that the incorporation of [3H]thymidine, another measure of cell proliferation, is increased in the human HT29 colon cancer cell line by 1009 –1007 M 9-cis-RA. In addition, the growth of the SW 948 human colon cancer cell line, as measured by cell number, is enhanced by micromolar concentrations of atRA and other synthetic retinoids [8]. The colon appears not to be the only tumor cell type whose growth can be stimulated by retinoids. While enhanced proliferation in the presence of retinoids is a rare occurrence, physiological concentrations (1008 M) of atRA enhance the growth of the prostatic cancer cell line LNCaP [2, 36]. The proliferation of GH-1 pituitary tumor cells [37] is also increased when these cells are exposed to atRA. This suggests that while retinoids may suppress the progression of some cancers, they could promote the growth of malignant cells found in the colon and other tissues. The RARs and RXRs are believed to mediate the majority of biological effects produced by retinoids. In addition to forming homodimers, RARs and RXRs can bind together to form heterodimers which can activate gene transcription [38–42]. Both atRA and 9-cis-RA can bind to RARs; the RXRs, however, have only been shown to bind 9-cis-RA. In transactivation assays measuring receptor function, 9-cis-RA acts as a full agonist and is equipotent in activating both RARs and RXRs. atRA is an ineffective agonist for RXRs in these assays; however, it acts as an agonist with an efficacy and potency comparable to 9-cis-RA for RARs [43]. If RXR homodimers were the only type of retinoid receptor complex involved in the proliferative effects of retinoids in MC-26 cells, one would expect 9-cis-RA, the naturally occurring RXR ligand, to be a more potent activator of cell growth than atRA. However, our data demonstrate that atRA and 9-cis-RA are equipotent in increasing MC-26 cell proliferation. Thus, the responses we have measured most likely involve activation of a RAR. Since DNA binding and gene activation by RARs is greatly enhanced and, in some cases, requires heterodimerization with an RXR [39, 41, 44, 45], our data do not eliminate the possibility that RXRs play a role in the growth stimulatory effects elicited by retinoids. Our data demonstrate that there are significant levels of RARa and RARg mRNA present in MC-26 cells, while RARb mRNA is expressed at very low levels. The
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very low to nonexistent basal expression of the RARb mRNA in the MC-26 cells has led us to hypothesize that the RARb may not play a significant role in retinoidinduced increases in MC-26 colon cancer cell growth. This hypothesis is supported by recent data which suggest the RARb is the direct mediator of the growth inhibitory effect produced by retinoids in breast cancer cells [4]. It may be that in cells where the RARb functions to regulate proliferative responses to retinoids, the net effect is growth inhibition. Like the RARb, the RARa has been associated with the growth inhibitory effects of retinoids in breast cancer cells [27, 28]. While the RARb plays a direct role in the growth inhibitory effect of retinoids, the RARa has been shown to modulate this response via regulation of RARb mRNA and protein levels [4]. To test whether the RARa is involved in the retinoid-induced increases in colon cancer cell growth, we examined the ability of an RARa-selective antagonist to inhibit the growth effect. This RARa antagonist, Ro 41-5253, has been shown in transactivation assays to selectively block biological effects mediated by the RARa, while having little effect on the RARb and RARg [29]. However, in MC-26 cells, Ro 41-5253 was unable to prevent the atRA-induced growth effect. These data suggest that the RARa is not involved in retinoid-induced increases in MC-26 colon cancer cell growth. It is possible that the RARg may mediate the growth effects of retinoids in the MC-26 cell line. We have been able to detect both mRNA and protein for the RARg within MC-26 cells. The lack of selective antagonists for the RARg, however, has prevented the further characterization of the involvement of RARg in the retinoidinduced increase in MC-26 cell proliferation at this time. Future experiments involving antisense oligonucleotides designed to deplete RARg levels may be useful in addressing the role of the RARg in retinoid-mediated increases in MC-26 cell growth. Although retinoids increased the proliferation of the MC-26 cell line, the growth of the LoVo cell line was not significantly modulated in the presence of retinoids. Therefore, our data demonstrate that growth stimulation is not produced by retinoids in every colon cancer cell line. Work done by Kane et al. [35] also showed that colon cancer cells can vary in their response to retinoids. In their studies, the proliferative rate of the human HT-29 colon cancer cell line was enhanced in the presence of 9-cis-RA. Caco-2 human colon cancer cells, however, responded to 9-cis-RA with a decrease in proliferation, as measured by [3H]thymidine incorporation. Additional data supporting the growth inhibitory effects of retinoids in Caco-2 cells have been obtained by McCormack et al., who have noted growth inhibition by 1008 M atRA after 3 days of retinoid exposure [46]. These studies provide further evidence that
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retinoids do not regulate the growth of all colon cancer cells in the same manner. The inability of the LoVo cells to respond to retinoids does not appear to be due to the absence of RARs within this cell line. LoVo cells possess the ability to synthesize the same retinoic acid receptor subtypes that are present in the MC-26 cell line. While both cell lines express some RARa, RARb, and RARg mRNA, there may be isoforms of these RAR subtypes present in MC26 cells which are absent in the LoVo cell line. Differences in RAR isoform expression could therefore explain the difference in retinoid responsiveness between the two cell lines. Another possibility is that the ability of retinoid receptors to interact with other signaling pathways could differ between the two colon cancer cell lines. Evidence supporting this hypothesis has been obtained in experiments performed by Rosewicz et al. [47] in human pancreatic carcinoma cells. This group was unable to detect retinoid receptor subtype differences between AsPc1 and Capan 2 cells, two pancreatic carcinoma cell lines which respond differently to atRA. They did, however, notice that in the AsPc1 cells, atRA induced expression of protein kinase Ca (PKCa), a protein kinase C subtype they have shown to be involved in the growth stimulation produced by atRA within this cell line [47]. Conversely, PKCa protein expression was decreased by atRA in the Capan 2 cells, whose growth is inhibited by atRA. The data collected by these authors therefore suggest that the net growth effect produced by retinoids in pancreatic carcinomas may be determined by the cell’s ability to modulate protein kinase C expression. In a likewise manner, retinoids in colon cancer cells may modulate the expression of protein kinases or other proteins which are required for retinoid-induced increases in cell growth. The growth response elicited by retinoids in colon cancer cells may therefore depend not only upon the retinoid receptor subtypes but also on what accessory proteins are expressed in the presence of retinoids. In summary, our data demonstrate that colon cancer cells differ in their ability to respond to both atRA and 9-cis-RA. The growth of LoVo cells are unaffected by the presence of retinoids, while the growth of MC-26 cells is enhanced following retinoid exposure. This variability suggests that retinoids may not be useful as therapeutic agents for colon cancer. Furthermore, care must be exercised when designing retinoid therapies for other types of cancers so that the growth of tumors that might be present in the colon will not be stimulated. We thank Drs. M. Klaus (Basel, Switzerland) and Jerry Sepinwall (Nutley, New Jersey) of Hoffman–LaRoche for their generous gift of the RARa antagonist Ro 41-5253 and 9-cis-retinoic acid, respectively.
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Received September 6, 1996 Revised version received January 13, 1997
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