Insulin modulates cellular proliferation in developing human jejunum and colon

Several lines of evidence suggest an important role for insulin in the regulatory mechanism of rodent small intestinal development. To investigate its potential implication in human gut, the immunofluorescent localization of insulin receptors (IR) and the influence of insulin (30 microU or 3 mU/ml) on [3H]-thymidine incorporation and on lactase and alkaline phosphatase activities were studied in fetal jejunum and colon (14-19 weeks). We demonstrate the early presence of IR, mainly detected in the basolateral portion of enterocytes and colonocytes along the crypt-villus axis. Insulin increased [3H]-thymidine incorporation as well as epithelial labeling indices in cultured explants from jejunum and colon without affecting enzymic activities. This study establishes, for the first time, that insulin stimulates proliferation of epithelial cells expressing IR in both segments without affecting brush border hydrolases in the developing human gut.     

Trafficking of newly synthesized surfactant protein A in isolated rat alveolar type II cells

We examined the synthesis, transport, and localization of surfactant protein A (SP-A) in primary cultures of alveolar type II cells. In type II cells maintained in culture for 6 h, 39% of the SP-A pool detected with an enzyme-linked immunosorbent assay (ELISA) was found in lamellar bodies (LBs). After 24 h in culture, 53% of the cellular SP-A pool was found in LBs. The absolute amount of SP-A in the LB compartment was almost identical at 6 and 24 h of culture. In contrast to the results obtained with ELISA, 35S labeling of newly synthesized SP-A revealed that less than 7% of the cellular SP-A pool was in LBs at either 6 or 24 h of culture. In the 6-h cultures, 17% of the total (i.e., cells and media) [35S]SP-A pool was extracellular. In the 24-h cultures, 70% of the [35S]SP-A pool was extracellular. The secretion of [35S]SP-A was blocked by brefeldin A at all times. When medium containing newly secreted [35S]SP-A was incubated with alveolar type II cells maintained in culture for 24 h, the protein was taken up and incorporated into the LB fraction. More than 80% of the internalized SP-A was associated with the LB compartment after a 6 h incubation. The uptake of [35S]SP-A was blocked at 4 degrees C and was promoted by addition of unlabeled SP-A at 37 degrees C. These findings support a pathway of extracellular routing of SP-A prior to its accumulation in LBs in cultured type II cells.     

Ambient pressure stimulates immortalized human aortic endothelial cells to increase DNA synthesis and matrix metalloproteinase 1 (tissue collagenase) production

In the present study, we investigated the effect of ambient pressure on [3H]-thymidine incorporation and on the production of matrix metalloproteinase 1 (tissue collagenase/proMMP-1) using human aortic endothelial cells immortalized with simian virus 40 (SE-1). Incubation of cells at ambient pressures of 50 and 100 mmHg for 24 h slightly increased [3H]-thymidine incorporation when directly compared with normal culture conditions. The amount of [3H]-thymidine incorporated in SE-1 reached a maximum at 150 mmHg, while a further increase in pressure to 200 mmHg decreased incorporation. The same ambient pressure slightly stimulated human aortic intimal smooth muscle cells (SMC) to increase [3H]-thymidine incorporation but not medial SMC. Immunoblot analysis also showed that ambient pressure, ranging from 50 to 200 mmHg, like 12-O-tetradecanoyl-phorbol-13-acetate stimulated SE-1 to produce proMMP-1, an effect not seen with either intimal or medial SMC. The amount of proMMP-1 produced also reached a maximum level at 150 mmHg. We postulate that human endothelial cells are ambient pressure sensitive and that relatively lower ambient pressures play an important role in the growth of endothelial cells, while higher pressures injure endothelial cells, resulting in the initiation of atherosclerosis. This cell line may prove useful in the investigation of both the physiological and pathological roles of blood pressure on endothelial cell function.     

Reduced tumorigenicity of cultured neuroblastoma cells after treatment in vitro with dexamethasone

The effect of dexamethasone on tumorigenicity of cultured neuroblastoma and on de novo synthesis of DNA and protein was determined. Within 12 hr dexamethasone caused a dose-dependent inhibition of [3H]-thymidine incorporation into DNA. Incorporation of [3H]-leucine into protein was not affected by dexamethasone. Neurite formation was interrupted by actinomycin D or cycloheximide. Cells treated with dexamethasone before inoculation into A/J mice produced fewer tumors with longer latent periods than controls. About 2.6 times as many neuroblastoma cells treated with 50 micrograms/ml dexamethasone for 4 days were required for tumor development in 50% of recipient animals as compared to controls. Reduced tumorigenicity was dependent upon the length of treatment and the concentration of dexamethasone used. Cortexolone did not mimic the effects of dexamethasone. If, instead of inoculation, cells were replated and grown without dexamethasone, cellular aggregations appeared among the cells cultured in the absence of dexamethasone. By autoradiography, replated cells previously treated with ethanol displayed uniform incorporation of [3H]-thymidine, whereas replated cells from dexamethasone-treated cultures exhibited no incorporation in differentiated cells. However, incorporation was noted among the clusters. We hypothesize that tumors arising after dexamethasone treatment may be due to the presence of an unresponsive subpopulation of cells.     

Production of basic fetoprotein in human peripheral lymphocytes during blastic transformation

To clarify the significance of basic fetoprotein (BFP) in lymphocytes, we investigated whether BFP is produced in lymphocytes during blastic transformation. Peripheral blood lymphocytes obtained from 14 adults were cultured under the stimulation of lectins. The concentration of BFP in the culture medium (extracellular BFP) was estimated serially. The incorporation of [6-3H]-thymidine was assayed simultaneously. The intracellular BFP was measured by dual flow cytometry for DNA and BFP. A lymph node was studied immunohistochemically. Serum BFP was measured in four cases of lymphocytic leukaemia. In two cases, dual staining was performed. The intracellular BFP of the mitogen-stimulated lymphocytes was increased within 24 h. The extracellular BFP was increased exponentially from 72 h. The extracellular BFP at 96 h did not correlate with the [3H]-thymidine incorporation. The intracellular BFP increase began in G1 phase. Immunostaining showed that the B cells also produced BFP. The serum BFP level in leukaemia was high in 1 of 4 cases and the leukaemic cells in two cases showed high intracellular BFP content. These observations indicate that BFP is produced in activated human lymphocytes and in lymphocytic leukaemic cells. The production of BFP during blastic transformation will be a useful new in vitro model for studying the biological role of BFP, and BFP labelling may offer some new possibilities for the study of lymphocytes.     

Stimulation of pancreatic growth by cholecystokinin is mediated by high affinity receptors on rat pancreatic acinar cells

Pancreatic acinar cells possess both high low affinity receptors for cholecystokinin. The cholecystokinin analog caerulein, which exerts a trophic effect on the rat pancreas, acts as an agonist at both types of receptors. In contrast, the synthetic analog CCK-JMV-180, which also acts as an agonist at high affinity receptors, opposes the action of caerulein on the low affinity receptors. We report that infusion of either caerulein or CCK-JMV-180 into rats increases [3H]-thymidine incorporation into pancreatic DNA and causes the pancreatic weight as well as content of DNA, RNA, and protein to increase. CCK-JMV-180 also stimulates in-vitro incorporation of [3H]-thymidine into DNA of cultured rat acini. The finding that both caerulein and CCK-JMV-180 exert the same trophic effect on pancreatic acinar cells indicates that this effect is mediated via high affinity acinar cell cholecystokinin receptors.     

No effect of kinins on DNA synthesis in LNCaP prostate cancer cells

1. Prostate has kininogenase activity and expresses members of the tissue kallikrein gene family. The present study examined the effect of exogenous and endogenous kinins on growth of LNCaP prostate adenocarcinoma cells. 2. Rate of DNA synthesis was measured by incorporation over 4 h of [3H]-thymidine into a TCA insoluble fraction of LNCaP cells that had been cultured for 24 h. 3. Increased [3H]-thymidine incorporation was seen in response to 10 nmol/L testosterone (+103 +/- 5 s.e.%), dihydrotestosterone (+113 +/- 14%) and R1881 (+64 +/- 10%) (P < or = 0.001; n = 4). 4. In contrast 0.05, 5 and 1000 nmol/L lysyl-bradykinin had no effect (15 +/- 4, 10 +/- 9 and 5 +/- 3 s.e.%, respectively; n = 7). Des-Arg9[Leu8]-bradykinin (a B1 receptor antagonist) and/or D-Arg-[Hyp3,Thi5,8,D-Phe7]-bradykinin (a B2 receptor antagonist), 1 nmol/L, and indomethacin, 5 mumol/L, also had little or no effect. 5. In conclusion, kallidin and endogenous kinins and prostaglandins have little or no effect on DNA synthesis and therefore on the growth of LNCaP cells in comparison to the two-fold stimulation produced by androgens.     

Serum-free media for culturing and serial-passaging of adult human retinal pigment epithelium

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm-2. Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9+/-2.7% and 47.1+/-4.1%, respectively) and DMEM with serum (87.2+/-5.6% and 52.9+/-10.5%, respectively) than DMEM without serum (59.2+/-5.6% and 33.1+/-6.9%, respectively; P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12+/-3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21+/-5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.     

The cellular response of lambs to Brucella ovis before and after birth

The immunological response of lambs to Brucella ovis before and after birth was investigated. The establishment of indwelling cannulas in the efferent prescapular lymphatic ducts of foetal lambs allowed continual monitoring of the immune response of a single lymph node. Foetal lambs in the last trimester of pregnancy were shown to mount a strong cell-mediated immune response to B. ovis. Lymphocytes from the challenged lymph node stimulated with B. ovis in vitro usually first reacted significantly and had highest [3H]-thymidine incorporation between 4 and 6 days after primary and secondary challenge, whereas, lymphocytes from the unchallenged node did not exhibit significant [3H]-thymidine incorporation until some 24 h later. Lymphocytes from these lambs challenged as foetuses still exhibited significant [3H]-thymidine incorporation in response to B. ovis for 4 to 5 months after birth. The proportion of surface immunoglobulin-positive cells in efferent prescapular lymph of unchallenged lambs ranged from 0.5 to 2.0% but after B. ovis challenge this proportion ranged from 2.7 to 8.7% between 4 to 6 days after challenge. By 9 to 12 days after challenge, the proportion had declined to pre-challenge values.     

Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts

SPARC (secreted protein, acidic and rich in cysteine), also known as osteonectin, is an extracellular Ca+2-binding glycoprotein that inhibits the incorporation of [3H]-thymidine and delays the onset of S-phase in synchronized cultures of bovine aortic endothelial (BAE) cells. This effect appears not to be dependent on the functional properties of SPARC associated with changes in cell shape or inhibition of cell spreading. In this study we investigate the conditions under which cell cycle modulation occurs in different types of cells. Human umbilical vein endothelial cells, a transformed fetal BAE cell line, and bovine capillary endothelial cells exhibited a sensitivity to SPARC and a cationic peptide from a non-Ca+2-binding region of SPARC (peptide 2.1, 0.2-0.8 mM) similar to that observed in BAE cells. In contrast, human foreskin fibroblasts and fetal bovine ligament fibroblasts exhibited an increase in the incorporation of [3H]-thymidine in the presence of 25 microM-0.2 mM peptide 2.1; inhibition was observed at concentrations in excess of 0.4 mM. This biphasic modulation could be further localized to a sequence of 10 amino acids comprising the N-terminal half of peptide 2.1. A synthetic peptide from another cationic region of SPARC (peptide 2.3) increased [3H]-thymidine incorporation by BAE cells and fibroblasts in a dose-dependent manner. In endothelial cells, a stimulation of 50% was observed at a concentration of 0.01 mM; fibroblasts required approximately 100-fold more peptide 2.3 for levels of stimulation comparable to those obtained in endothelial cells. The observation that SPARC and unique SPARC peptides can differentially influence the growth of fibroblasts and endothelial cells in a concentration-dependent manner suggests that SPARC might regulate proliferation of specific cells during wound repair and remodeling.     

Altered levels of histone H1(0) and DNA topoisomerase activity in the liver of the tumour-bearing rat

Walker 256 carcinoma cells were injected subcutaneously into the dorsal region of rats. After 9 days of tumour growth increases were observed in liver mass and DNA content and in the incorporation of [3H]-thymidine into liver DNA. These changes were accompanied by a fall in the H1(0) fraction of liver histones and rises in both DNA topoisomerase I and II activities.     

Intracranial aneurysms treated with the Guglielmi detachable coil: midterm clinical results in a consecutive series of 100 patients

The purpose of this study was to investigate in vitro the potential effect of type 1 collagen gel containing alpha-elastin on the proliferation of vascular smooth muscle cells and vascular endothelial cells, and on smooth muscle cell migration. Vascular smooth muscle cell and endothelial cell were cultured in 12-well plates precoated with collagen gels and alpha-elastin. Cell proliferation rates were measured by monitoring [3H]-thymidine incorporation. After 2, 3 or 4 days of culture, the proliferation rate of both smooth muscle cells and endothelial cells was significantly decreased on collagen gel containing 10 mg/ml alpha-elastin compared with collagen gel only as control. Smooth muscle cell proliferation on collagen gel containing alpha-elastin on the 4th day of culture was decreased dose-dependently, e.g. 1 mg/ml of alpha-elastin (74.8(2.3)% of control, P=n.s.); 5 mg/ml (56.7(2.1)%; P<0.05); 10 mg/ml (30.3(3.1)%; P<0.005). In the case of cultured endothelial cells, however, [3H]-thymidine incorporation was not decreased significantly in the presence of 5 mg/ml alpha-elastin (83.1(7.9)%, P=n.s.). After stimulation by platelet-derived growth factor, the smooth muscle cell migration rate on collagen gel containing alpha-elastin (5 mg/ml) was decreased over time. The area of migration on the 6th day of culture was also significantly decreased dose-dependently in the presence of alpha-elastin, e.g. 1 mg/ml (72.6(3.4)% of control, P<0.05), 5 mg/ml (56.9%(1.5)%; P<0.05); 10 mg/ml (37.3(2.7)%; P<0.0005). In conclusion, alpha-elastin inhibited the proliferation and migration of smooth muscle cell in a dose-dependent manner on collagen gel culture, however, at high concentrations of alpha-elastin (10 mg/ml), the endothelial cell proliferation rate was also inhibited. At 5 mg/ml, alpha-elastin significantly inhibited smooth muscle cell proliferation and migration but did not significantly inhibit endothelial cell proliferation. Incorporation of collagen gel containing alpha-elastin into the structure of arterial prosthesis offers the possibility of inhibiting smooth muscle cell hyperplasia without significant effect on endothelial cell formation.     

Patient doses in abdominal aortogram and aorta femoral runoff examinations

BACKGROUND: The present study was designed to assess the effects of insulin-like growth factor-I (IGF-I) alone and in combination with growth hormone (GH) on differentiation and replication in cultured human granulosa cells. METHODS: Granulosa cells from patients undergoing in vitro fertilization were isolated and cultured for 2 days in culture medium with 10% serum, the medium was removed and the cells were incubated with IGF-I (1, 10 and 100 ng/ml) with or without GH (10 ng/ml) in serum-free medium and in the presence of 3H-methylthymidine (2 microCi/ml). RESULTS: IGF-I alone resulted in a significant dose-dependent increase in medium estradiol (E2) (p<0.05) and progesterone (P) (p<0.001) and suppression of IGF-binding protein-1 (IGFBP-1) (p<0.001), without any increase in [3H]-thymidine incorporation (P=0.10). The combination of IGF-I and GH further increased the release of E2 (p<0.001), and the amount of [3H]-thymidine incorporation (p<0.001). CONCLUSION: These results indicate a synergistic effect of IGF-I and GH on differentiation and replication of human granulosa cells, and thus support a role of both GH and IGF-I in regulation of ovarian function.     

Hyperproliferation of aortic smooth muscle cells and fibroblasts from young SHR rats is not shared by endothelial cells

1. To study the hypertensive genotypic influence on growth kinetics of the three aortic wall cell types. 2. Using young spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats weighing 160-180 g, we compared the proliferative properties of endothelial cells (EC), smooth muscle cells (SMC) and fibroblasts that were isolated from the thoracic aorta of each strain and cultured. Growth-arrested cells were exposed to P < -thymidine after stimulation with 150 micrograms/mL endothelial cell growth supplement. Proliferation assays were performed by cell seeding on decellularized aortic explants and cell counting 2, 4, 5, 6 and 7 days after seeding. The influence of SMC from SHR on the growth kinetics of EC was evaluated by co-cultures in transwell systems. 3. After stimulation, SMC from SHR exhibited a greater P < -thymidine incorporation rate than those from WKY rats (ratios over controls: 3.90 +/- 0.48 [7] vs 1.85 +/- 0.25 [7] respectively, P < 0.05). This was also true for adventitial SHR fibroblasts: (13.1 +/- 0.6 [6] vs 9.9 +/- 1.0 [6] WKY P < 0.05). On the contrary, there was no difference in the P < -thymidine incorporation rates between EC of each strain, regardless of the passage and the time considered. Cell proliferation on matrix explants confirmed the hyperproliferation of SMC and fibroblasts from SHR, while EC of each strain proliferated equally. Smooth muscle cells from SHR did not influence the growth kinetics of EC in co-culture and vice versa. 4. The changes in growth patterns of aortic cells isolated from young prehypertensive SHR seem to be restricted to SMC and fibroblasts.     

Activation of deoxycytidine kinase during inhibition of DNA synthesis by 2-chloro-2'-deoxyadenosine (Cladribine) in human lymphocytes

Deoxycytidine kinase (dCK, EC.2.7.1.74), a key enzyme in intracellular metabolism of many antileukemic drugs, was shown to be activated during treatment of lymphocytes by 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine), a potent inhibitor of DNA synthesis. While 5-[3H]-thymidine (TdR) incorporation into DNA was decreased by 80-90%, dCK activity was doubled as a consequence of incubating the cells with 1 microM 2-chloro-2'-deoxyadenosine. Thymidine kinase (dTK, EC.2.7.1.21) activity was slightly decreased under the same conditions, similarly to 5-[3H]-thymidine incorporation. dCK activation could not be prevented by cycloheximide, and neither the amount of dCK protein nor its mRNA level was increased after 2-chloro-2'-deoxyadenosine treatment. These results suggest a post-translational activation of dCK protein during inhibition of DNA synthesis.     

Angiotensin II potentiates DNA synthesis in AT-1 transformed cardiomyocytes

Angiotensin II has been shown to be mitogenic in various cell types. In cultured neonatal cardiomyocytes, we have demonstrated that angiotensin II causes hypertrophy, not hyperplasia. However, fetal or neonatal cardiomyocytes exhibit limited proliferation in primary culture, and are mitotically less potent. In order to determine whether angiotensin II is simply a hypertrophic or hyperplastic growth factor for mitotically-potent cardiomyocytes, we analysed [3H]-thymidine uptake and cell cycle-regulated gene expression using SV40 large T-transformed AT-1 cardiomyocytes. Angiotensin II, alone and in combination with other growth factors, increased [3H]-thymidine uptake in a dose-dependent manner. The mRNA expression of G1 cyclins (Cyclin C, D1, D2, D3) and histone H1-kinase activity by CDK2 increased 6 h after angiotensin II stimulation. Western blot analysis revealed cyclin B1 expression after 18 h , which peaked at 30 h. Histone H1-kinase activity by cdc2 was also increased by angiotensin II, and peaked at 24-36 h, indicating that these changes were cell cycle dependent. Double immunofluorescent photography showed that AT-1 cells incorporated BrdU, and expressed cdc2 by angiotensin II stimulation. [3H]-thymidine and BrdU uptake were blocked by losartan, but not by PD123319. In contrast with neonatal cardiomyocytes, angiotensin II potentiated DNA synthesis and induced cell cycle regulated gene expression in AT-1 cardiomyocytes, and this activity was mediated by the angiotensin II type-1 receptor.     

Ascorbate affects proliferation of guinea-pig vascular smooth muscle cells by direct and extracellular matrix-mediated effects

Proliferation of smooth muscle cells and deposition of extracellular matrix proteins are important events in the formation of atherosclerotic plaques. We have investigated the direct and matrix-mediated effects of ascorbate on the proliferation rate of vascular smooth muscle cells (VSMC) isolated from the guinea-pig aorta. In the presence of ascorbate, cells showed a bi-phasic growth pattern. At 125 microM ascorbate, -3H--thymidine incorporation was stimulated 25%. However, higher concentrations of ascorbate gradually decreased cell-incorporated radioactivity up to 50% at 2 mM ascorbate. These effects of ascorbate on DNA synthesis in VSMC were paralleled by the changes in cell number and were not due to ascorbate cytotoxicity. Alpha-tocopherol (0.1 mM), individually and in combinations with 1 mm ascorbate, also inhibited DNA synthesis in VSMC. Ascorbate also influenced proliferation of smooth muscle cells through matrix-mediated effect. New VSMC culture plated on extracellular matrices deposited by smooth muscle cells in the presence of 0.1-1 mM ascorbate had up to 50% lower proliferation rate than on matrices from ascorbate-deficient cells, as assessed by [3H]-thymidine incorporation. This effect was independent from alpha-tocopherol and specific inhibitors of collagen synthesis: L-azetidine-2-carboxylic acid and pyridine-2,4-dicarboxylic acid. An ascorbate-dependent matrix effect was specific for smooth muscle cells grown on VSMC and human skin fibroblast-originated matrices, but not for human vascular endothelial cells. The possible involvement of ascorbate in the regulation of smooth muscle cells proliferation by its antioxidant/pro-oxidant effects and regulation of extracellular matrix composition are discussed.     

Cyclooxygenase inhibition reveals synergistic action of vasoconstrictors on mesangial cell growth

Since endogenous vasoconstrictors promote mesangial cell growth and increase the biosynthesis of antiproliferative prostaglandins, the effects of cyclooxygenase inhibition on mesangial cell proliferation should be strongly dependent on the prevailing levels of neuroendocrine vasoconstrictors. We compared the effects of indomethacin (10(-6) M), a cyclooxygenase inhibitor, on [3H]thymidine incorporation by cultured rat mesangial cells in the presence of various combinations of angiotensin II (10(-10) M), [Arg8]vasopressin (10(-11) M), (-)-norepinephrine (10(-8) M) and endothelin-1 (10(-11) M). Indomethacin did not enhance [3H]thymidine incorporation in cells treated with each individual vasoconstrictor, or in cells treated with two-way combinations with the exception of modestly increased [3H]thymidine incorporation in cells treated with angiotensin II + (-)-norepinephrine or [Arg8]vasopressin + (-)-norepinephrine. In contrast, in cells treated with any three-way or the four-way combination, indomethacin markedly increased [3H]thymidine incorporation. Importantly, a highly significant interaction (P<0.0001) was observed for thymidine incorporation between the number of vasoconstrictors present and indomethacin treatment, thus demonstrating that cyclooxygenase inhibition reveals a synergistic action of vasoconstrictors on the DNA synthesis in mesangial cells.     

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.