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U.S. Department of Agriculture, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202-9034
2To whom correspondence should be addressed. E-mail: hzeng{at}gfhnrc.ars.usda.gov.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • human fecal water • selenium • cell cycle • colon cell
The colonic epithelium is in an equilibrium between cell proliferation and cell death. Alterations in the control of cell cycle progression and programmed cell death (apoptosis) have been implicated in many human diseases including cancer (1). Colon cancer results from the accumulation of mutations in certain genes that control cell cycle, apoptosis and DNA repair (2). The association between colon cancer and diet has been well established in human epidemiologic studies as well as in animal studies (3,4).
Recently, there has been considerable interest in the role of the aqueous phase of human feces (fecal water) in studies examining the mechanisms underlying the dietary etiology of colon cancer (5–13) because components of this fecal fraction are in direct contact with colonic epithelial cells. Dietary factors have been shown to significantly affect the cytotoxicity and genotoxicity of fecal water (6–13). This cytotoxicity has been hypothesized to cause epithelial cell loss in the large bowel, resulting in increased cell proliferation, which may be linked to a higher risk for the development of colon cancer (14,15). A preliminary study suggested that human fecal water fractions could induce apoptosis in HT-29 colon adenocarcinoma cells (16). Furthermore, components of fecal water have been shown to alter specific genes that control the cell cycle, apoptosis and DNA repair (11,12), thus suggesting that some components of fecal water might be protective against cancer.
Selenium, an essential trace element for humans, can either stimulate or inhibit cell growth depending on its concentration and chemical form (17). At concentrations greater than nutritional requirements, selenium has either anticancer effects through cell cycle arrest and apoptosis pathways or toxic effects through cell necrosis pathways (18,19). The alteration of cell cycle progression and apoptosis triggered by selenium has been implicated as an important mechanism for its anticancer effects in mammary and prostate cells (20–22). Because selenium is not absorbed completely, it is likely that the element is present in the hindgut, particularly when high doses are consumed. However, only a few studies have looked at the effect of selenium on cell cycle progression in colonic cells (23,24).
In view of the putative role of alterations in cell cycle progression in colon cells during tumor development, it is important to understand the mechanisms by which human fecal water and selenium can affect this outcome. Because of the remarkable efficacy of sodium selenite in inducing cell growth arrest and showing cancer prevention in other cell types, sodium selenite was included as a reference for comparison in the present study. The following studies were conducted to investigate the molecular basis of the effect of human fecal water and selenite on HT-29 cell cycle progression.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Sodium selenite, dithiothreitol and propidium iodide (PI) were purchased from Sigma Chemical (St. Louis, MO). Cytidine 5'-triphosphate (
-32P) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Oligonucleotides were synthesized by Gibco BRL (Rockville, MD).
Fecal water collection.
The study participants were the same 20 healthy men (including 3 individuals who attended only the equilibration period) from the previous study (13). Fecal water from all 20 men was prepared by modifying a published procedure (6) during the 1-wk equilibration period. As reported, during that period, they consumed a controlled mixed Western diet with adequacy in all known nutrients (13). Briefly, fecal samples were stored at 4°C for <12 h, and homogenized with a Masticator homogenizer (IUL Instruments, Barcelona, Spain) for 2 min; fecal water was prepared by centrifuging the sample for 2 h at 30,000 x g at 4°C. The supernatant was decanted and centrifuged again at 30,000 x g for 15 min at 4°C. The supernatant was then filtered through a 149-µm Nitex filter, and the samples were stored at -80°C. The procedure does not involve further isolation of the fractions of different components in fecal water, and should reflect the intact free water in the stool. To decrease the day-to-day variability among samples, individual composites for each subject were made from all of the fecal water. To minimize the subject-to-subject variability among samples, three composite pools from 3 subjects, 8 subjects and 9 subjects, respectively, were made and used in this study. Before cell treatment, fecal water was filtered through a 0.2-µm sterile filter (Pall, Ann Arbor, MI).
Cell culture and cell growth.
HT-29 cells were maintained in DMEM media (GIBCO-BRL, Grand Island, NY) with 10% (v/v) fetal bovine serum (FBS) in a humidified atmosphere 95% air/5% CO2 at 37°C, and cell passage was between 133 and 150. Cells (3 mL) were seeded at 1.5–2 x 108 cells/L into each well of a 6-well plate. After 24 h, human fecal water, selenite or an equal volume of PBS buffer was added to study the effect on cell cycle/growth. The concentration of human fecal water in DMEM media was measured as a percentage (v/v). Cells were counted by a hemocytometer. Cells that excluded trypan blue after incubation with an equal volume of PBS containing 4 g/L trypan blue dye were considered viable.
Cell cycle analysis.
Cell cycle was analyzed by using flow cytometry with PI staining. HT-29 cells were tryspinized and washed once with PBS and incubated in 70% (v/v) ethanol at -20°C. After the incubation, cells were washed with PBS and stained with 50 mg PI/L with 6000 U RNase A/L. The DNA contents of cells were determined by flow cytometry. Data were stored as list-mode files of at least 10,000 single-cell events and analyzed by EPICS profile II and ModFit LT software (Coulter, Miami, FL and Topsham, ME).
Gene array and RT-PCR assay.
Total cellular RNA was isolated from HT-29 cells by using an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, and the integrity of RNA samples was checked by electrophoresis. Each cDNA probe was prepared from 5 µg of total cellular RNA, and hybridized to Human Cell cycle-1 GEArray membrane (Superarray, Bethesda, MD). These membranes contain 23 sequence-verified known cell cycle–related genes. Each cell cycle–related gene was normalized by 10% of the ß-actin signal in the same membrane, and only those gene signals (
10% of the ß-actin signal) that were well above the background were considered specific gene signals. These data were collected, stored and analyzed with Molecular Dynamics Image-Quant system (Sunnyvale, CA). To confirm the data generated by gene array analysis, an RT-PCR (20–25 cycles) (Promega, Madison, WI) assay was performed (25) with the ß-actin gene as an internal control. Briefly, three independent RNA samples were isolated from HT-29 cells, and RNA template samples were serially diluted to make sure that RT-PCR products were within a linear range. Therefore, the intensity of RT-PCR products directly correlated with the mRNA level of proliferating cell nuclear antigen (PCNA), gadd45 or p21 gene. The primer pairs were as follows: ß-actin primers: 5'-ATG GGT CAG AAG GAT TCC TAT G-3'; 5'-CAG CTC GTA GCT CTT CTC CA-3; PCNA primers: 5'-CCA TCC TCA AGA AGG TGT TGG-3'; 5'-GTG TCC CAT ATC CGC AAT TTT AT-3'; DNA damage–inducible transcript 1 (Gadd45) primers: 5'-GCA ATA TGA CTT TGG AGG AAT TC-3'; 5'-CCA TCA CCG TTC AGG GAG ATT-3'; p21 primers: 5'-ACT GTG ATG CGC TAA TGG CG-3'; 5'-TTT GAG GCC CTC GCG CTT C-3'.
Analysis of PCNA promoter activity and transient transfection.
A fragment of genomic DNA containing the 5'-flanking sequence, first exon and partial first intron (26) was generated by Pfu enzyme (Stratagene, La Jolla, CA) with primers 5'-TAC GAG CGC ATC AAT TCT GTA AT-3; 5'-CCA ACA GGT TTA GTG AGC AAA GA-3'. The 1121-bp PCR fragments were cloned into the polylinker (SmaI site) of the pGL3 reporter vector (Promega), and the orientation was confirmed by obtaining the predicted fragment size after PCR with insert and vector primers. Cells were transiently transfected with PCNA promoter construct, pRL-null vector (Promega), OPTI-MEM I and lipofectamine reagent for 4 h and then placed in culture media for 16 h before being exposed to human fecal water or selenite for 24 h. The cells were lysed and assayed for reporter gene activity using the Luciferase Assay Reagent according to the manufacturer’s instructions (Promega). Luciferase activity was measured in a luminometer (Turner Designs, Sunnyvale, CA). The expression levels of the luciferase reporter protein in the transfected cells were normalized in reference to Renilla luciferase activity in the same cells.
Statistical analysis.
Results are given as means ± SEM. Statistical analyses were performed by one-way ANOVA followed by Dunnett’s multiple comparisons to the control group. Differences with a P-value < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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There was a decrease in the growth of HT-29 cells treated with human fecal water or sodium selenite (Fig. 1). Interestingly, the G1 phase cell distribution increased, and the S + G2 phase cell distribution decreased in cells treated with fecal water (Fig. 2; Table 1). In contrast, the S + G2 phase cell distribution increased and the G1 phase cell distribution decreased in selenite-treated cells (Fig. 2; Table 1).
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Gene array analysis showed that the mRNAs were expressed at different levels in human fecal water and selenite-treated cells. The mRNA levels of cyclin A and PCNA were decreased by 69 and 62%, respectively, in 5% human fecal water treated HT-29 cells. Cyclin A mRNA was not affected and PCNA mRNA was decreased only 28% in selenite-treated HT-29 cells. The mRNA level of gadd45 was increased 2.28-fold in 4 µmol/L sodium selenite–treated HT-29 cells but was not changed in cells treated with human fecal water (Table 2). These results were confirmed by RT-PCR. A weaker mRNA signal of PCNA was seen in HT-29 cells treated with 5% human fecal water compared with control cells treated with PBS buffer (Fig. 3). On the other hand, gadd45 mRNA signals were stronger in HT-29 cells treated with 4 µmol/L sodium selenite than in control cells treated with PBS buffer (Fig. 3). In contrast, p21 mRNA signals were much stronger in HT-29 cells treated with 5% human fecal water or 4 µmol/L sodium selenite than in control cells treated with PBS buffer (Fig. 3).
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With the promoter sequence fused to the reporter plasmid in the correct or reverse (R) orientation, reporter constructs were transiently transfected into HT-29 cells. The correct orientation plasmid displayed four- to fivefold higher activity than the reverse orientation construct in HT-29 cells (Fig. 4). This indicated that not only had we succeeded in isolating the promoter region of the PCNA gene, but also that the promoter was active in HT-29 cells. Because HT-29 cells become fragile after DNA transfection, we examined the effect of human fecal water and sodium selenite at concentrations < 5% and 4 µmol/L, respectively, to avoid the induction of cell death. Human fecal water (1.25 or 2.5%) dramatically decreased the activity of the PCNA promoter in a dose-dependent manner (Fig. 4). In contrast, sodium selenite (2 or 3 µmol/L) caused only minor decreases in the PCNA gene promoter activity and the response was not dose dependent.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The gene expression of cell cycle–related genes in cells treated with fecal water and selenite was determined via gene array analysis followed by conventional RT-PCR validation of the genes with differential expression >2.0. Our data demonstrated that both human fecal water (5%) and 4 µmol/L selenite greatly increased the mRNA level of the cyclin-dependent kinase inhibitor gene, p21waf1. Interestingly, the mRNA levels of cyclin A and PCNA were dramatically decreased in HT-29 cells treated with fecal water but not selenite. In agreement with the PCNA mRNA levels, human fecal water but not sodium selenite significantly decreased PCNA promoter activity in a dose-dependent manner. In contrast, the mRNA level of the gadd45 was significantly increased in HT-29 cells treated with selenite but not fecal water.
These differential changes observed in the expression of key cell cycle regulatory genes suggest several important molecular effects of human fecal water and selenite on colonic cell cycle progression. First, eukaryotic cell cycle progression is orchestrated by cyclin-dependent kinases (CDK) and proteins that regulate CDK (27,28). The deregulation of their gene expression is directly related to colon cancer risk (2,27). The induction of the p21waf1 is a common mechanism of growth arrest in different physiologic situations (25). p21waf1 consists of at least two functional domains that bind to PCNA and Cdk/cyclins (29). It has been shown that p21waf1 inhibits PCNA-dependent DNA replication by preventing PCNA from contributing to DNA polymerase 
and 
function; it induces G2/M arrest after DNA damage (29) and leads to efficient G1 arrest (30). It is thus conceivable that the up-regulation of p21waf1 mRNA in cells treated with 5% human fecal water or 4 µmol/L sodium selenite is responsible in part for the cell growth arrest in G1 and S + G2-mol/L phase transition, respectively. Second, cyclin A associates not only with cdk1, but also with cdk2, and it is clear that degradation of cyclin A during mitosis is critical for G1 cell cycle arrest (31). Furthermore, the role of PCNA in cell cycle control is related to its interaction with cyclin and CDK (30,32). Therefore, the down-regulation of cyclin A and PCNA mRNAs in cells treated with human fecal water indicates that fecal water negatively affected cyclin A and PCNA gene transcription, consistent with the previous observation of G1 cell cycle arrest (31). In contrast, although sodium selenite also inhibited HT-29 cell cycle progression, it did not greatly affect PCNA and cyclin A mRNA expression. Third, the growth arrest and DNA damage-inducible gene (gadd45) is associated with cell growth inhibition, DNA damage response and DNA repair (33). In agreement with previous findings (34), we observed that sodium selenite induced gadd45 gene transcription. Previous studies have shown that gadd45 interacts with PCNA as well as with the universal cyclin-dependent kinase inhibitor p21, and this interaction is critical for negative growth control (35). In contrast to selenite, human fecal water shows little effect on gadd45 gene expression, suggesting that direct DNA damage is likely responsible in part for selenite but not for cell cycle arrest induced by fecal water.
It is possible that the study of PCNA promoter activity in HT-29 cells exposed to fecal water from humans fed different experimental diets may be utilized as a biomarker of the effect of diet on colon cancer susceptibility. Several reports indicate that the cytotoxicity and genotoxicity of human fecal water are dependent on dietary factors (6–13). Therefore, it is likely that dietary factors will determine the effect of human fecal water on PCNA promoter activity. In the future, human fecal water obtained from people fed different experimental diets may be used to assess PCNA promoter activity as a potential biomarker of cancer protective components in the fecal water. Because colon cancer risks are affected by numerous dietary factors, it is conceivable that many other fecal water–sensitive genes would be available for future investigation.
In summary, both human fecal water and sodium selenite inhibited HT-29 cell cycle progression. In contrast to the S and G2 cell cycle arrest induced by sodium selenite, human fecal water caused G1 cell cycle arrest. Although both human fecal water and sodium selenite increased the mRNA level of p21waf1, the up-regulation of the gadd45 gene by sodium selenite and the down-regulation of PCNA and cyclin A genes by human fecal water represented distinct mechanisms of cell cycle arrest. Furthermore, the finding that human fecal water inhibited PCNA gene promoter activity in a dose-dependent manner suggests a novel approach for assessing the effect of diet on colon cancer susceptibility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Present address: National Institutes of Health/NCI, Nutritional Science Research Group, Rockville, MD 20892-7328.
4 Abbreviations used: CDK, cyclin-dependent kinase; FBS, fetal bovine serum; gadd45, DNA damage–inducible transcript 1; PCNA, proliferating cell nuclear antigen; PI, propidium iodide; p21waf1, cyclin-dependent kinase inhibitor 1A.
Manuscript received 23 February 2003. Initial review completed 18 April 2003. Revision accepted 28 May 2003.
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LITERATURE CITED |
|---|
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Thompson, C. B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science (Washington, DC) 267:1456-1462.
2. Kinzler, K. W. & Vogelstein, B. (1996) Lessons from hereditary colorectal cancer. Cell 87:157-170.
3. Potter, J. D. (1996) Colon cancer—do the nutritional epidemiology, the gut physiology and the molecular biology tell the same story?. J. Nutr. 123(suppl. 2):418-423.
4. Davis, C. D., Zeng, H. & Finley, J. W. (2002) Selenium-enriched broccoli decreases intestinal tumorigenesis in multiple intestinal neoplasia mice. J. Nutr. 132:307-309.
5. Rafter, J., Child, P., Anderson, A. M., Alder, R., Eng, V. & Bruce, W. R. (1987) Cellular toxicity of fecal water depends on diet. Am. J. Clin. Nutr. 45:559-563.
6. Glinghammar, B., Venturi, M., Roland, I. R. & Rafter, J. J. (1997) Shift from a dairy product-risk to a dairy product-free diet: influence on cytotoxicity and genotoxicity of fecal water—potential risk factors for colon cancer. Am. J. Clin. Nutr. 66:1277-1282.
7. Lapre, J. A., De Vries, H. T. & Van Der Meer, R. (1993) Cytotoxicity of fecal water is dependent on the type of dietary fat and is reduced by supplemental calcium phosphate in rats. J. Nutr. 123:578-585.
8. De Kok, T. M., Van Faassen, A., Glinghammar, B., Pachen, D. M., Eng, M., Rafter, J. J., Baeten, C. G., Engels, L. G. & Kleinjans, J. C. (1999) Bile acid concentrations, cytotoxicity and pH of fecal water from patients with colorectal adenomas. Dig. Dis. Sci. 44:2218-2225.[Medline]
9. Rieger, M. A., Parlesak, A., Pool-Zobel, B. L., Rechkemmer, G. & Bode, C. (1999) A diet high in fat and meat but low in dietary fibre increases the genotoxic potential of "faecal water.". Carcinogenesis 20:2311-2316.
10. Lapre, J. A. & Van der Meer, R. (1992) Diet-induced increase of colonic bile acids stimulates lytic activity of fecal water and proliferation of colonic cells. Carcinogenesis 13:41-44.
11. Glinghammar, B., Holmberg, K. & Rafter, J. (1999) Effects of colonic lumenal components on AP-1-dependent gene transcription in cultured colon carcinoma cells. Carcinogenesis 20:969-976.
12. Glinghammar, B. & Rafter, J. (2001) Colonic luminal contents induce cyclooxygenase 2 transcription in human colon carcinoma cells. Gastroenterology 120:401-410.[Medline]
13. Davis, C. D. (2003) Low dietary copper increases fecal free radical production, fecal water alkaline phosphatase activity and cytotoxicity in healthy men. J. Nutr. 133:522-527.
14. Terpstra, O. T., Van Blankenstein, M., Dees, J. & Eilers, G. (1987) Abnormal cell proliferation in the entire colonic mucosa of patients with colon adenomas or cancer. Gastroenterology 92:704-708.[Medline]
15. Stadler, J., Yeung, K. S., Furrer, R., Marcon, N. & Himaal, H. S. (1988) Proliferative activity of rectal mucosa and soluble fecal bile acids in patients with normal colons and patients with colonic polyps or cancer. Cancer Lett. 38:315-320.[Medline]
16. Haza, A. I., Glinghammar, B., Grandien, A. & Rafter, J. (2000) Effect of colonic luminal components on induction of apoptosis in human colonic cell lines. Nutr. Cancer. 36:79-89.[Medline]
17. Ip, C., Thompson, H. J. & Ganther, H. E. (2000) Selenium modulation of cell proliferation and cell cycle biomarkers in normal and premalignant cells of the rat mammary gland. Cancer Epidemiol. Biomark. Prev. 9:49-54.
18. Fleming, J., Ghose, A. & Harrison, P. R. (2001) Molecular mechanisms of cancer prevention by selenium compounds. Nutr. Cancer. 40:42-49.[Medline]
19. Zeng, H. (2001) Arsenic suppresses necrosis induced by selenite in human leukemia HL-60 cells. Biol. Trace Elem. Res. 83:1-15.[Medline]
20. Wang, Z., Jinag, C. & Lu, J. (2002) Induction of caspase-mediated apoptosis and cell-cycle G1 arrest by selenium metabolite methylselenol. Mol. Carcinog. 34:113-120.[Medline]
21. Venkateswaran, V., Klotz, L. H. & Fleshner, N. E. (2002) Selenium modulation of cell proliferation and cell cycle biomarkers in human prostate carcinoma cell lines. Cancer Res. 62:2540-2545.
22. Sinha, R., Unni, E., Ganther, H. E. & Medina, D. (2001) Methylseleninic acid, a potent, growth inhibitor of synchronized mouse mammary epithelial tumor cells in vitro. Biochem. Pharmacol. 61:311-317.[Medline]
23. Baines, A., Taylor-Parker, M., Goulet, A. C., Renaud, C., Gerner, E. W. & Nelson, M. A. (2002) Selenomethionine inhibits growth and suppresses cyclooxygenase-2 (COX-2) protein expression in human colon cancer cells. Cancer Biol. Ther. 4:370-374.
24. Chigbrow, M. & Nelson, M. (2001) Inhibition of mitotic cyclin B and cdc2 kinase activity by selenomethionine in synchronized colon cancer cells. Anticancer Drugs. 12:43-50.[Medline]
25. Chang, B. D., Watanable, K., Broude, E. V., Fang, J., Poole, J. C., Kalinichenko, T. V. & Roninson, I. B. (2000) Effects of p21Waf1/Cip1/Sdi on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases. Proc. Natl. Acad. Sci. U.S.A. 97:4291-4296.
26. Almendral, J. M., Huebsch, D., Blundell, P. A., Macdonald-Bravo, H. & Bravo, R. (1987) Cloning and sequence of the human nuclear protein cyclin: homology with DNA-binding proteins. Proc. Natl. Acad. Sci. U.S.A. 84:1575-1579.
27. Pines, J. (1995) Cyclins, CDKs and cancer. Semin. Cancer Biol. 6:63-72.[Medline]
28. Grana, X. & Reddy, E. P. (1995) Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 20:211-219.
29. Dulic, V., Stein, G. H., Far, D. F. & Reed, S. I. (1998) Nuclear Accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition. Mol. Cell. Biol. 18:546-557.
30. Niculescu, A. B., Chen, X., Smeets, M., Hengst, L., Prives, C. & Reed, S. I. (1998) Effects of p21Cip1/waf1 at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Mol. Cell. Biol. 18:629-643.
31. Jacobs, H. W., Keidel, E. & Lehner, C. F. (2001) A complex degradation signal in Cyclin A required for G1 arrest, and a C-terminal region for mitosis. EMBO. J. 20:2376-2386.[Medline]
32. Prosperi, E. (1997) Multiple roles of the proliferating cell nuclear antigen: DNA replication, repair and cell cycle control. Prog. Cell Cycle Res. 3:193-210.[Medline]
33. Fornace, A. J., Jr, Alamo, I., Jr & Hollander, M. C. (1988) DNA damage-inducible transcripts in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 85:8800-8804.
34. Kaeck, M., Lu, J., Strange, R., Ip, C., Ganther, H. E. & Thompson, H. J. (1997) Differential induction of growth arrest inducible genes by selenium compounds. Biochem. Pharmacol. 53:921-926.[Medline]
35. Vairapandi, M., Azam, N., Balliet, A. G., Hoffman, B. & Liebermann, D. A. (2000) Characterization of MyD118, Gadd45, and Proliferating cell nuclear antigen (PCNA) interacting domains. J. Biol. Chem. 275:16810-16819.
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