Biomedical subjects

R D Beauchamp

Publications and source records attributed to R D Beauchamp.

At least 37 records · Page 2Linked to original sources

PubMed1999

Optimal interpretation of FDG PET in the diagnosis, staging and management of pancreatic carcinoma.

UNLABELLED: This study had two purposes: to optimize the semiquantitative interpretation of 18F-fluorodeoxyglucose (FDG) PET scans in the diagnosis of pancreatic carcinoma by analyzing different cutoff levels for the standardized uptake value (SUV), with and without correction for serum glucose level (SUV(gluc)); and to evaluate the usefulness of FDG PET when used in addition to CT for the staging and management of patients with pancreatic cancer. METHODS: Sixty-five patients who presented with suspected pancreatic carcinoma underwent whole-body FDG PET in addition to CT imaging. The PET images were analyzed visually and semiquantitatively using the SUV and SUV(gluc). The final diagnosis was obtained by pathologic (n = 56) or clinical and radiologic follow-up (n = 9). The performance of CT and PET at different cutoff levels of SUV was determined, and the impact of FDG PET in addition to CT on patient management was reviewed retrospectively. RESULTS: Fifty-two patients had proven pancreatic carcinoma, whereas 13 had benign lesions, including chronic pancreatitis (n = 10), benign biliary stricture (n = 1), pancreatic complex cyst (n = 1) and no pancreatic pathology (n = 1). Areas under receiver operating characteristic curves were not significantly different for SUV and SUV(gluc). Using a cutoff level of 3.0 for the SUV, FDG PET had higher sensitivity and specificity than CT in correctly diagnosing pancreatic carcinoma (92% and 85% versus 65% and 61%). There were 2 false-positive PET (chronic pancreatitis, also false-positive with CT) and 4 false-negative PET (all with true-positive CT, abnormal but nondiagnostic) examinations. There were 5 false-positive CT (4 chronic pancreatitis and 1 pancreatic cyst) and 18 false-negative CT (all with true-positive FDG PET scans) examinations. FDG PET clarified indeterminate hepatic lesions or identified additional distant metastases (or both) in 7 patients compared with CT. Overall, FDG PET altered the management of 28 of 65 patients (43%). CONCLUSION: FDG PET is more accurate than CT in the detection of primary tumors and in the clarification and identification of hepatic and distant metastases. The optimal cutoff value of FDG uptake to differentiate benign from malignant pancreatic lesions was 2.0. Correction for serum glucose did not significantly improve the accuracy of FDG PET. Although FDG PET cannot replace CT in defining local tumor extension, the application of FDG PET in addition to CT alters the management in up to 43% of patients with suspected pancreatic cancer.

Adenocarcinoma↗

PubMed1998

Induction of cyclooxygenase-2 by activated Ha-ras oncogene in Rat-1 fibroblasts and the role of mitogen-activated protein kinase pathway.

Elevated cyclooxygenase-2 (COX-2) expression and activity have been observed in several different transformed cell types that express mutated ras genes. To investigate the mechanism of increased COX-2 expression following Ras-mediated transformation, Rat-1:iRas cell line was transfected with an Ha-RasVal-12 cDNA expression vector that is under the transcriptional control of the lac operon and is inducible with isopropyl-1-thio-beta-D-galactopyranoside (IPTG). IPTG treatment caused parallel increases in the levels of Ha-Ras and COX-2 proteins in Rat-1:iRas cells. The increased expression of COX-2 was accompanied by increased prostaglandin E2 production. Selective inhibition of COX-2 activity suppressed the production of prostaglandin E2 by >90% but did not alter the progress of the morphological transformation. The level of COX-2 mRNA was up-regulated by activated Ha-Ras. Induction of Ras increased the transcription of COX-2 by 44.3 +/- 10.1% and increased the half-life of COX-2 mRNA by approximately 3.5-fold. A specific mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) inhibitor (PD 98059) caused a delay in both the activation of ERK1/2 and the induction of COX-2 in IPTG-induced Rat-1:iRas cells. Inhibition of ERK activity by PD 98059 also suppressed the induction of COX-2 by epidermal growth factor in intestinal epithelial cells and significantly reduced the expression of COX-2 in Ha-Ras-transformed rat intestinal epithelial cells. ERK activity appears to be required for induction of COX-2 by Ras.

Animals↗

PubMed1998

TGF-beta1 effects on proliferation of rat intestinal epithelial cells are due to inhibition of cyclin D1 expression.

Transforming growth factor-beta 1 (TGF-beta1) arrests intestinal epithelial cells (RIE-1 and IEC-6) in the G1 phase of the cell cycle and inhibits cyclin D1 expression. This report describes experiments designed to elucidate the mechanism of cyclin D1 inhibition and to determine whether inhibition of cyclin D1 expression is the cause, rather than the result, of TGF-beta1-mediated cell cycle arrest. TGF-beta1 inhibition of IEC-6 cell proliferation was associated with a decrease in the abundance of cyclin D1/Cdk4 complexes and a corresponding decrease in Cdk4-dependent phosphorylation of the retinoblastoma protein. Metabolic labeling studies indicated that TGF-beta1 inhibited cyclin D1 synthesis without altering the rate of cyclin D1 protein degradation. Cyclin D1 antisense oligonucleotides blocked serum-stimulated induction of cyclin D1 and DNA synthesis, whereas cyclin D1 sense oligonucleotides had no effect. RIE-1 cells were engineered to overexpress human cyclin D1 under the control of a tetracycline-repressible promoter. These cells entered S phase in the presence of TGF-beta1 only when human cyclin D1 was derepressed by the withdrawal of tetracycline. These data indicate that TGF-beta1 inhibits the synthesis of cyclin D1 in gut epithelial cells and that this inhibition is the cause, rather than the result, of TGF-beta1-mediated arrest of intestinal epithelial cell proliferation.

Animals↗

PubMed1998

Antioxidants reduce cyclooxygenase-2 expression, prostaglandin production, and proliferation in colorectal cancer cells.

Increased expression of cyclooxygenase (COX) and overproduction of prostaglandins (PGs) have been implicated in the development and progression of colorectal cancer (CRC). Recent observations suggest that reactive oxygen intermediates play a role in tumor cell growth regulation and expression of the inducible COX, COX-2. We therefore evaluated the effects of various antioxidants on COX expression and cellular growth in the human CRC cell line HCA-7. The antioxidants pyrrolidinedithiocarbamate (PDTC), N-acetylcysteine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and U74006 decreased PG production, intracellular redox status, and cellular growth in a concentration-dependent manner. The decrease in cellular growth was associated with the induction of apoptosis. Unlike the selective COX inhibitors 1-[(4-methylsulfonyl)phenyl]-3-trifluoromethyl-5-[(4-fluoro)phenyl]pyraz ole (SC 58125) and (2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS 398) that inhibit COX-2 catalytic activity, these antioxidants decreased COX-2 expression at the transcriptional level. Combined treatment of HCA-7 cells with PDTC and SC 58125 resulted in an additive decrease in PG levels and anchorage-dependent and -independent growth. Furthermore, whereas antioxidants or SC 58125 reduced tumor growth in vivo, coadministration of PDTC and SC 58125 resulted in actual tumor regression. These results suggest that combined therapy with NSAIDs and antioxidants might be useful in the prevention and/or treatment of CRC.

Animals↗

PubMed1998

Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells.

Previously, we have shown that forced expression of prostaglandin endoperoxide synthase-2 [also called cyclooxygenase (COX) 2] leads to inhibition of programmed cell death in intestinal epithelial cells. More recently, we have demonstrated that growth of human colonic cancer xenografts is inhibited by treatment with a highly selective COX-2 inhibitor in tumors that express COX-2 (HCA-7) but not in those that lack COX-2 expression (HCT-116). To explore the biochemical mechanisms involved in these effects, we have evaluated the role of COX-2-derived eicosanoid products on programmed cell death in human colon cancer cells. Here we report that PGE2 treatment of human colon cancer cells leads to increased clonogenicity of HCA-7, but not HCT-116 cells. Treatment with a highly selective COX-2 inhibitor (SC-58125) decreases colony formation in monolayer culture and this growth inhibition was reversed by treatment with PGE2. Additionally, PGE2 inhibits programmed cell death caused by SC-58125 and induces Bcl-2 expression, but did not affect Bcl-x or Bax expression in human colon cancer (HCA-7) cells. Therefore, decreased cell death caused by PGE2 would enhance the tumorigenic potential of intestinal epithelial cells. Thus, these results may help to explain a component of the mechanism by which COX inhibitors prevent colorectal cancer in humans.

Animals↗

PubMed1998

Nuclear translocation of beta-catenin in hereditary and carcinogen-induced intestinal adenomas.

The physical interaction between beta-catenin and the adenomatous polyposis coli (APC) gene, and the ability of APC to regulate cytoplasmic levels of beta-catenin suggest a role for beta-catenin in colorectal carcinogenesis. In this study, we found that beta-catenin immunoreactivity was detected exclusively in the cell membrane and cytoplasm of morphologically normal intestinal epithelial cells with predominant distribution in the differentiated nonproliferative cell population. In contrast, beta-catenin was localized predominantly in the nucleus of adenomas from Min/+ mice and transgenic mice expressing a mutant truncated form of the APC gene (Apc(delta716) mice). Beta-catenin was expressed predominantly at the cell membrane and cytoplasm of the nontransformed rat intestinal epithelial (RIE-1) cells in culture, whereas predominantly nuclear localization of beta-catenin was observed in the human colon cancer cell line SW480. In the azoxymethane (AOM) treated rats, overexpression and nuclear localization of beta-catenin was observed in all adenomas. Previous studies have indicated the incidence of APC mutations amongst AOM-induced tumors to be 15% or less. These results demonstrate that nuclear localization of beta-catenin is a common event in colorectal tumorigenesis.

Adenoma↗

PubMed1997

Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2.

A considerable amount of evidence collected from several different experimental systems indicates that cyclooxygenase-2 (COX-2) may play a role in colorectal tumorigenesis. Large epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in persons taking aspirin or other nonsteroidal antiinflammatory drugs on a regular basis. One property shared by all of these drugs is their ability to inhibit COX, a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been characterized, COX-1 and COX-2. COX-2 is expressed at high levels in intestinal tumors in humans and rodents. In this study, we selected two transformed human colon cancer cell lines for studies on the role of COX-2 in intestinal tumorigenesis. We evaluated HCA-7 cells which express high levels of COX-2 protein constitutively and HCT-116 cells which lack COX-2 protein. Treatment of nude mice implanted with HCA-7 cells with a selective COX-2 inhibitor (SC-58125), reduced tumor formation by 85-90%. SC-58125 also inhibited colony formation of cultured HCA-7 cells. Conversely, SC-58125 had no effect on HCT-116 implants in nude mice or colony formation in culture. Here we provide evidence that there may be a direct link between inhibition of intestinal cancer growth and selective inhibition of the COX-2 pathway.

Animals↗

PubMed1997

Decreased transforming growth factor beta type II receptor expression in intestinal adenomas from Min/+ mice is associated with increased cyclin D1 and cyclin-dependent kinase 4 expression.

Tumor cells often become resistant to the growth-inhibitory effects of transforming growth factor beta (TGF-beta). Recent studies have identified TGF-beta type II receptor (RII) mutations in a subset of cancers, including colon cancer. To evaluate the expression of TGF-beta RII in premalignant intestinal adenomas and the relationship with cell cycle regulation, we investigated the expression of TGF-beta RII, cyclin D1, and cyclin-dependent kinase 4 (Cdk4) in Min/+ mouse intestinal adenomas. Immunohistochemistry indicated that TGF-beta RII cytoplasmic immunoreactivity was undetectable in the proliferative crypt zones of the normal small intestinal and normal colonic epithelium but was abundant toward the villus tips of the normal small intestine and the lumenal third of the colonic glands. As was observed in the proliferating crypt zones, TGF-beta RII immunoreactivity was dramatically decreased or undetectable in all adenomas examined in comparison to the abundant levels in adjacent normal differentiated intestinal epithelium. TGF-beta RII mRNA was also reduced in the adenomas in comparison to normal mucosa as determined by reverse transcription-PCR. In an inverse distribution to TGF-beta RII, Cdk4 nuclear immunoreactivity was restricted to the crypt regions of the small and large intestine, whereas cyclin D1 immunoreactivity was uniformly absent in normal intestinal epithelium. For both cyclin D1 and Cdk4, protein and mRNA levels were increased in intestinal adenomas but not in normal intestinal epithelium as determined by immunohistochemistry, in situ hybridization, and reverse transcription-PCR. In summary, the lack of TGF-beta RII expression was associated with increased cyclin D1 and Cdk4 expression in Min/+ mouse intestinal adenomas. We hypothesize that the former may enable tumor cells to escape from the normal growth-constraining influence of TGF-beta, whereas the latter promotes inappropriate cell proliferation and adenoma progression.

Adenoma↗

PubMed1997

Concurrent overexpression of cyclin D1 and cyclin-dependent kinase 4 (Cdk4) in intestinal adenomas from multiple intestinal neoplasia (Min) mice and human familial adenomatous polyposis patients.

We postulated that increased expression of the cell cycle regulators cyclin D1 and cyclin-dependent kinase (Cdk) 4 may be involved in the development of intestinal adenomas associated with familial adenomatous polyposis (FAP). In the present study of multiple intestinal neoplasia (Min) mice and human FAP patients, the expression and distribution of cyclin D1, Cdk4, and cell proliferative activity (5-bromo-2'-deoxyuridine incorporation) in normal and adenomatous intestinal epithelium were investigated. Immunohistochemical analysis of Min mouse intestine revealed that cyclin D1 immunoreactivity in the intestinal epithelium was restricted to the adenomatous areas, with a significantly higher percentage of positively staining nuclei in high-grade dysplasia versus low-grade dysplasia (54.8 +/- 18.4% versus 34.6 +/- 16.9%, P = 0.016). Morphologically normal areas of intestinal epithelia were uniformly negative for cyclin D1 immunoreactivity. Cdk4 nuclear immunoreactivity was restricted to the crypt areas in morphologically normal small intestine and colon. Conversely, Cdk4 immunoreactivity was uniformly abundant in adenomatous areas regardless of the degree of dysplasia. Increased expression of cyclin D1 and Cdk4 in adenomas was accompanied by a significantly increased 5-bromo-2'-deoxyuridine incorporation rate in the same areas. Immunoblot analysis of lysates from surgical specimens revealed increased levels of cyclin D1 and Cdk4 in the majority of intestinal adenomas from human FAP patients in comparison to the adjacent grossly normal colonic mucosa. Our results indicate that overexpression of cyclin D1 and Cdk4 occurs in intestinal adenomas and is associated with increased cell proliferative activity in premalignant neoplastic cells. Increased cyclin D1 immunoreactivity is associated with more severe dysplasia. These data suggest that abnormal up-regulation of these important G1 cell cycle proteins is a relatively early event in intestinal carcinogenesis and that these changes may contribute to malignant progression within those lesions.

Adenomatous Polyposis Coli↗

PubMed1997

A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells.

BACKGROUND & AIMS: Constitutive expression of cyclooxygenase 2 (COX-2) has been found in 85% of colorectal cancers. Ras mutations are found in 50% of colorectal adenocarcinomas. The aim of this study was to determine the role of COX-2 in ras-induced transformation in rat intestinal epithelial (RIE) cells. METHODS: Cell growth was determined by cell counts. The expression of COX-2 was examined by Northern and Western analyses. For tumorigenicity assays, cells were inoculated into dorsal subcutaneous tissue of athymic nude mice. DNA-fragmentation assays were performed to detect apoptosis. RESULTS: The expression of COX-2 was increased in RIE-Ras cells at both messenger RNA (9-fold) and protein (12-fold) levels. Prostaglandin I2 levels were elevated 2.15-fold in RIE-Ras cells. Serum deprivation further increased COX-2 expression 3.8-fold in RIE-Ras cells. Treatment with a selective COX-2 antagonist (SC58125) inhibited the growth of RIE-Ras cells through inhibition of cell proliferation and by induction of apoptosis. SC-58125 treatment reduced the colony formation in Matrigel by 83.0%. Intraperitoneal administration of SC-58125 suppressed RIE-Ras tumor growth in nude mice by 60.3% in 4 weeks. SC-58125 treatment also induced apoptosis in RIE-Ras cells as indicated by increased DNA fragmentation. CONCLUSIONS: Overexpression of COX-2 may contribute to tumorigenicity of ras-transformed intestinal epithelial cells. Selective inhibition of COX-2 activity inhibits growth of ras-transformed intestinal epithelial cells and induces apoptosis.

Animals↗

PubMed1997

Antioxidants enhance the cytotoxicity of chemotherapeutic agents in colorectal cancer: a p53-independent induction of p21WAF1/CIP1 via C/EBPbeta.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Five-fluorouracil (5FU) remains the single most effective treatment for advanced disease, despite a response rate of only 20%. Herein, we show that the antioxidants pyrrolidinedithiocarbamate and vitamin E induce apoptosis in CRC cells. This effect is mediated by induction of p21WAF1/CIP1, a powerful inhibitor of the cell cycle, through a mechanism involving C/EBPbeta (a member of the CCAAT/enhancer binding protein family of transcription factors), independent of p53. Antioxidants significantly enhance CRC tumor growth inhibition by cytotoxic chemotherapy in vitro (5FU and doxorubicin) and in vivo (5FU). Thus, chemotherapeutic agents administered in the presence of antioxidants may provide a novel therapy for colorectal cancer.

Acetylcysteine↗

PubMed1997

Cyclooxygenase-2 induction and transforming growth factor beta growth inhibition in rat intestinal epithelial cells.

Rat intestinal epithelial cells (RIE-1) permanently transfected with the prostaglandin endoperoxide synthase 2 (also referred to as cyclooxygenase-2; COX-2) gene exhibit decreased cyclin D1 levels, decreased cdk4-associated kinase activity, and delayed G1 cell cycle progression, which represents a phenotype similar to that which follows transforming growth factor beta (TGF-beta) treatment. In the current study, we have found that addition of TGF-beta 1 to the parental RIE-1 cells (designated RIE-P) caused a rapid induction of COX-2 mRNA and protein. COX-2 protein levels progressively increased and reached peak levels 6 h after TGF-beta 1 addition. Cyclin D1 was decreased by 74% at 6 h and was undetectable 24 h after addition of TGF-beta 1. In RIE cells transfected with the COX-2 antisense expression vector (RIE-AS cells), TGF-beta 1 induction of COX-2 protein was reduced greater than 90%. Addition of TGF-beta 1 did not reduce the abundant cyclin D1 protein expression in the RIE-AS cells, unlike the effect in RIE-P cells. TGF-beta 1 treatment reduced peak [3H]thymidine incorporation by 60% and delayed G1/S-phase transition by at least 4 h in the RIE-P cells. In contrast, S-phase entry occurred at 16 h in RIE-AS cells and was not altered by TGF-beta 1 treatment. Restoration of cyclin D1 expression by transfection of the cyclin D1 cDNA under transcriptional control of the cytomegalovirus promoter/enhancer in the COX-2-overexpressing (RIE-S) cells decreased the time required for S-phase entry by at least 4 h and increased the peak level of [3H]thymidine incorporation. Taken together, the results demonstrate that TGF-beta 1 strongly induces COX-2 at both the mRNA and protein levels and suggest that this induction of COX-2 is involved in the down-regulation of cyclin D1 and inhibition of cell growth caused by TGF-beta 1 in rat intestinal epithelial cells.

Animals↗

PubMed1996

Short-term caloric restriction augments age-related decreases in gastrin content and release.

Aging is associated with significant structural and functional changes in the gastrointestinal tract. Gastrin, a hormone produced by G cells in the antrum of the stomach, stimulates proliferation of gastric mucosa; its synthesis appears to decrease with age. Life-long restriction of caloric intake is the only experimental manipulation that has been shown to retard aging processes in rats. The purpose of this study was to examine the effect of short-term caloric restriction (CR) on the production and release of the hormone gastrin with aging. Aging causes a fall in both fasting plasma levels of gastrin and antral content of gastrin in Fischer 344 rats; short-term CR appears to augment this age-related decrease. Steady state levels of antral gastrin mRNA were decreased with aging, and short-term CR resulted in an augmented decrease in aged, but not in young rats. Our findings indicate that gastrin release, synthesis and gene expression decrease with age. Restriction of the caloric intake for a short period (i.e. 8 weeks) augments this age-related decrease in antral gastrin and fasting plasma levels. Short-term CR appears to decrease the production of gastrin at the level of gene expression.

Aging↗

PubMed1996

G1 delay in cells overexpressing prostaglandin endoperoxide synthase-2.

Colorectal cancer is the second leading cause of death from cancer in the United States. Continuous use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to reduce the risk of colorectal cancer in humans by 40-50%. Patients with familial adenomatous polyposis who take NSAIDs, such as sulindac, undergo a regression of intestinal adenomas. Rodents exposed to carcinogens that cause colon cancer have a 50-60% reduction in the size and number of colonic tumors when treated continuously with NSAIDs. One common target for these drugs is prostaglandin endoperoxide synthase, also referred to as cyclooxygenase (COX). We and others have shown recently that COX-2 levels are increased dramatically in 85-90% of human colorectal adenocarcinomas and in 40-50% of colonic adenomas. We prepared intestinal epithelial cells that express the COX-2 gene permanently and found that they have altered adhesion properties and resist undergoing apoptosis. We report here that these cells also have a 3-fold increase in the duration of G1, lower levels of cyclin D1 protein, and a marked decrease in retinoblastoma kinase activity associated with cyclin-dependent kinase 4. The delay in G1 transit may relate to the resistance of these cells to undergo programmed cell death, which could affect their tumorigenic potential.

Adenoma↗

PubMed1996

Mechanisms of bombesin on growth of gastrinoma (PT) in vivo.

The growth of the human gastrinoma model (PT) in athymic nude mice is stimulated by bombesin (BBS), an amphibian peptide homologous to both human gastrin-releasing peptide (GRP) and neuromedin B (NMB). The mechanism is not known, and a potent and specific GRP-R antagonist BIM26226, which has low affinity for NMB-R, was used in vivo in athymic nude mice bearing gastrinoma subcutaneously. Both the BBS and BIM26226 stimulated the growth of PT, and the growth stimulation was even greater when given together. RT-PCR study of gastrinoma revealed the presence of both GRP-R and NMB-R mRNA, but much more abundant NMB-R mRNA. We conclude that BBS-stimulated growth of gastrinoma involves both GRP-R and NMB-R, and our findings suggest that GRP-R mediates negative and NMB-R produces positive growth effects on gastrinoma.

Animals↗

PubMed1996

Elevated cyclooxygenase-2 levels in Min mouse adenomas.

BACKGROUND & AIMS: Mutations in the APC gene result in an increased propensity to develop intestinal neoplasia; however, a complete understanding of the mechanisms resulting in tumor formation has remained elusive. Min mice possess a mutation in the APC gene and display a neoplastic phenotype similar to that observed in familial adenomatous polyposis coli in humans. Cyclooxygenase (COX) inhibitors decrease tumor multiplicity in the Min mouse intestine. The present study was designed to determine if there was an increase in COX-2 in adenomas harvested from Min mouse intestine. METHODS: COX-2 messenger RNA levels were determined by Northern blots and reverse-transcription polymerase chain reactions of B6Min x 129 mouse-derived tumors. Protein levels and localization were determined by Western blots and immunohistochemical staining. RESULTS: The Northern blots revealed an approximately threefold increase in the level of COX-2 messenger RNA in Min mouse adenoma compared with normal mucosa. COX-2 protein levels in adenomatous tissues were also approximately threefold higher compared with normal mucosa from the same mouse. Immunohistochemical staining with a monospecific COX-2 antibody confirmed that increases in COX-2 immunoreactivity were restricted to dysplastic and neoplastic foci within intestinal mucosa. CONCLUSIONS: These data show that COX-2 levels may be increased at an early stage in colorectal neoplasia during polyp formation and before invasion.

Adenoma↗

PubMed1996

Intestinal cell cycle regulations. Interactions of cyclin D1, Cdk4, and p21Cip1.

OBJECTIVE: The p21Cip1 protein is a potent stoichiometric inhibitor of cyclin-dependent kinase activity, and p21Cip1 mRNA expression is localized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (IECs) underwent reversible cell cycle arrest by contact inhibition, and determined whether increases in the relative amount of p21 associated with cyclin D/Cdk4 protein complexes were associated with cell growth arrest. METHODS: Density arrest was achieved by prolonged culture IEC-6 in confluent conditions (5 or more days). Release from density arrest was achieved by detaching the cells from the culture plate and reseeding them at a 1:4 ratio. The DNA synthesis was estimated by [3H]-thymidine incorporation and expressed as mean plus or minus standard error of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein levels were determined by standard Northern and Western blot analyses, respectively. Cyclin D1, Cdk4, and p21 protein complex formation was analyzed by immunoprecipitating the complexes from cell lysates with an antibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitated complexes using antibodies to the other proteins. The kinase activity of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. RESULTS: The IEC-6[3H]-thymidine incorporation was decreased 7.5-fold from day 1 confluence to day 7 of confluence. Twenty-four hours after release from density arrest, there was a 43-fold increase in [3H]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immunoblotting showed that the levels of cyclin D1 and Cdk4 proteins decreased by 70.9% and 68.7%, respectively, comparing day 3 with day 9 during density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-arrested cultures, coincident with the increase in DNA synthesis. The amount of p21 associated with the cyclin D1 and Cdk4 complex in the density-arrested cells was 170% of that observed in the reseeded, proliferating cells. More important, the p21::Cdk4 ratio was 6.4-fold higher in the density-arrested (quiescent) cells as compared with rapidly proliferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release from growth arrest, coincident with decreased binding of p21 to the complex. CONCLUSIONS: Intestinal epithelial cells in culture can undergo density-dependent growth arrest. This process involves downregulation of cyclin D1 and Cdk4 at the level of protein expression, whereas the mRNA levels remain relatively unchanged. Further, during contact inhibition, there is more p21 associated with cyclin D1/Cdk4, which further contributes to the inhibition of the kinase complex. The authors also have shown that the process of contact inhibition is reversible, which may explain partly the ability of the intestinal epithelium to increase proliferative activity in response to injury.

Animals↗

PubMed1995

Transforming growth factor-beta 1 inhibits cyclin D1 expression in intestinal epithelial cells.

Transforming growth factor-beta 1 (TGF-beta 1) inhibits most epithelial cell types by blocking cell cycle progression during the G1 phase. D cyclins are normally expressed during G1 and are regulators of G1 progression. One of the crucial functions of D cyclins is their ability to bind to a cyclin-dependent kinase (Cdk4). In mink lung epithelial cells, TGF-beta 1 inhibits Cdk4 expression. We have measured cell cycle progression and D cyclins and Cdk4 expression in non-transformed rat intestinal epithelial cell lines (IEC-6 and RIE-1) after TGF-beta 1 treatment. In exponentially growing cultures, TGF-beta 1 blocked DNA synthesis and suppressed cyclin D1 mRNA and protein expression, whereas the levels of cyclins D2, D3 and Cdk4 remained relatively unchanged. TGF-beta 1 was also added to G0-synchronized IEC-6 cells after serum stimulation. TGF-beta 1 prevention of G1 progression was associated with an inhibition of cyclin D1 protein expression. Cyclin D3 levels were not affected by TGF-beta 1 during G1 traverse. Our results suggest that cyclin D/Cdk4 is a crucial target of TGF-beta 1 and that regulation of this kinase is mediated through cyclin D1 in intestinal epithelial cells.

Animals↗