Differential effects of transforming growth factor-beta and epidermal growth factor on the cell cycle of cultured rabbit articular chondrocytes

We examined the effect of transforming growth factor (TGF-beta) on the proliferative rate and cell cycle of cultured rabbit articular chondrocytes using cell counting, cytofluorometry, and [3H]-thymidine incorporation. In the presence of 2% or 10% FCS (fetal calf serum), TGF-beta at 0.01, 0.1, 1, and 10 ng/ml had an inhibitory effect on cell proliferation after 24 h exposure with a dose dependence only for 2% FCS. Flow cytometric analysis of cell DNA content at that time showed that a high proportion of cells were arrested in late S-phase (SQ or G2Q) in either 2% or 10% FCS-containing medium. In both cases, a disappearance of the cell blockage occurred between 24 and 48 h after TGF-beta addition. However, whereas a stimulation of cell proliferation rate was observed at that time in cultures containing 10% FCS, a dose-dependence inhibition of cell growth was detected, in contrast, for 2% FCS-treated cells. Presence of TGF-beta during the last 24 h was not necessary to release the arrested cells. Furthermore, platelet-poor plasma at 10% produced the same effects as FCS, suggesting that platelet-derived factors, such as platelet-derived growth factor (PDGF), could not be responsible for the release of blocked cells in this case. We compared the effect of TGF-beta to that of epidermal growth factor (EGF), used at an optimal concentration (10 ng/ml). In both slowly growing (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. No arrest in late S-phase but an augmentation of the percentage of cells in S- and G2M-phases were observed. When combined, TGF-beta and EGF did not induce synergistic effect on the chondrocyte proliferation, as estimated by cell counting. [3H]-thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF-beta than for EGF (approximately 6 h versus 12 h, respectively). Although both factors induce similar cell-number increases at 48 h in 10% FCS-containing medium, these proliferative effects were due to different actions on the cell cycle. The present study indicates that TGF-beta induces first a recruitment of chondrocytes in noncycling SQ- or G2Q-blocked cells. The, the release of these cells may produce either apparent stimulation of cell proliferation if sufficient levels of an unknown serum factor are present (10% FCS) or an inhibition of growth rate when only reduced amounts of this factor are available (2% FCS).(ABSTRACT TRUNCATED AT 400 WORDS)     

TGF-beta-induced G2/M delay in proliferating rabbit articular chondrocytes is associated with an enhancement of replication rate and a cAMP decrease: possible involvement of pertussis toxin-sensitive pathway.

This study was undertaken to gain more insight into the mechanism whereby TGF-beta influences the cell cycle progression of cultured rabbit articular chondrocytes. Using proliferating chondrocytes in fetal calf serum-containing medium, we have previously shown that TGF-beta induced a recruitment of cells at the end of the S phase (G2/M) observed 24 h after addition. The delayed cells may then be released, producing a proliferative effect at 48 h, provided a substantial amount of FCS (10%) is present in the medium. Otherwise, in low level of serum (2% FCS, for example), only inhibition of cell proliferation is observed. In chondrocytes synchronized in S phase by a thymidine block, we investigated here the time-course incorporation of [3H]-thymidine into DNA, the cell cycle traverse by flow cytofluorometric study of DNA content, the expression of PCNA (Proliferating Cell Nuclear Antigen), and cAMP levels. The data demonstrate that TGF-beta provoked a decrease of cAMP content (0.5-1 h) followed by an enhancement of the DNA synthesis rate (4 h) which was detectable through cytofluorometric analysis and [3H]-thymidine labeling and correlated with the PCNA expression. In contrast, addition of cAMP analogues to the cultures resulted in an inhibition of replication rate. We also showed that pertussis toxin produced a decrease of the DNA synthesis rate, in a transient manner and only in the presence of TGF-beta. All these results suggest that TGF-beta may accelerate the replication process of cyclized chondrocytes, making then accumulate at the G2/M boundary, via a mechanism that could involve the adenylate cyclase activity and a Gi-protein. The factor might be responsible for producing a pool of cells having already replicated their DNA and therefore capable of re-entering the cell cycle without delay. This cell population could serve as a tissue reserve able to induce a mitosis wave when necessary--for example, in the repair of tissue damage.     

Human purified interleukin-1 inhibits DNA synthesis and cell growth of osteoblastic cell line (MC3T3-E1), but enhances alkaline phosphatase activity in the cells

We examined the effects of human purified interleukin-1 (IL-1) on DNA synthesis, cell growth, and alkaline phosphatase activity in the osteoblastic cell line MC3T3-E1 under both preconfluent and confluent culture conditions. Addition of IL-1 to the cells markedly inhibited their DNA synthesis and growth over the range 1-10 U/ml. Such significant inhibitory effects were observed in cells cultivated in 1 or 5% fetal calf serum (FCS)-containing alpha modification Eagle's medium (alpha-MEM), but not in alpha-MEM containing 10% FCS. In contrast, alkaline phosphatase activity was enhanced significantly by IL-1 in the cell line cultivated in 1% FCS-containing alpha-MEM. These results demonstrate that human purified IL-1 is effective in inducing the differentiation of osteoblastic cell MC3T3-E1.     

Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.     

Δ2-Troglitazone promotes cytostatic rather than pro-apoptotic effects in breast cancer cells cultured in high serum conditions

We have previously shown that Δ2-Troglitazone (Δ2-TGZ) displayed anticancer effects on breast cancer cell lines grown in low serum conditions (1% fetal calf serum (FCS)). The present study was performed in order to characterize the effects of Δ2-TGZ in high serum containing medium and to determine if starvation could influence the response of breast cancer cells to this compound, keeping in mind the potential interest for breast cancer therapy. We observed that in high serum conditions (10% FCS), a 48 h treatment with Δ2-TGZ induced a decrease in cell numbers in MDA-MB-231 and MCF-7 breast cancer cell lines. The IC₅₀ values were higher than in low serum conditions. Furthermore, in contrast to our previous results obtained in 1% FCS conditions, we observed that in 10% FCS-containing medium, MCF-7 cells were more sensitive to Δ2-TGZ than MDA-MB-231 cells. Δ2-TGZ also induced endoplasmic reticulum (ER) stress mainly in MDA-MB-231 cells. Besides, in high serum conditions, Δ2-TGZ induced a G₀/G₁ cell cycle arrest, an inhibition of BrdU incorporation and a reduced level of cyclin D1. We observed a limited cleavage of PARP and a limited proportion of cells in sub-G₁ phase. Thus, in high serum conditions, Δ2-TGZ displayed cytostatic effects rather than apoptosis as previously reported in 1% FCS-containing medium. Our results are in accordance with studies suggesting that serum starvation could potentiate the action of diverse anti-cancer agents.     

Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3-E1) by epidermal growth factor

Prostaglandin (PG) E2, known as a bone-resorption factor, was released as a predominant arachidonate metabolite in the culture medium of an osteoblastic cell line cloned from mouse calvaria (MC3T3-E1). Epidermal growth factor (EGF) (10 ng/ml) prominently enhanced endogenous PGE2 synthesis, requiring the simultaneous presence of unidentified factor(s) contained in bovine serum. PGE2 synthesis increased after a lag phase for 1-2 h and reached a maximum level at about 3 h after EGF addition. EGF-stimulated PGE2 synthesis was almost completely blocked by 10 microM cycloheximide or 1 microM actinomycin D. Furthermore, when the cells were pretreated with EGF, the microsomes exhibited an increased activity of fatty acid cyclooxygenase (arachidonic acid----PGH2), whereas the activity of PGE synthase (PGH2----PGE2) remained unchanged. These results suggested an EGF-mediated induction of cyclooxygenase. Following increased PGE2 synthesis, DNA synthesis increased and alkaline phosphatase activity decreased in a slower response to EGF. PGE2 (above 0.1 microM) added to the cells could replace EGF. However, such effects of EGF on the osteoblasts could not be attributed totally to an autocrine function of PGE2 produced by stimulation with EGF because these effects of EGF were not abolished by indomethacin, which blocked the PGE2 synthesis.     

Modulation of the mitogenic response of an epidermal growth factor-dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor-beta

The stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGR receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up-regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high-affinity receptors (less than 500) are required for EGF-dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF-stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin-like growth factors I or II (IGF-I, IGF-II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF-I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor-beta (TGF-beta) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF-beta receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF-beta) significantly altered 125I-EGF binding characteristics in MK cells, suggesting a point of action distal to 125I-EGF binding.     

Transforming growth factor beta (TGF-beta) inhibits hepatocyte DNA synthesis independently of EGF binding and EGF receptor autophosphorylation

Subpicomolar concentrations of human platelet-derived transforming growth factor beta (TGF-beta) inhibited growth factor-stimulated DNA synthesis in primary cultures of adult rat hepatocytes. This inhibition was not the result of changes in the size of intracellular pools of 3H-thymidine and was not dependent on the state of confluence of the cells. A 24-hr exposure to TGF-beta either before or after insulin/EGF stimulation was as inhibitory on DNA synthesis between 48 and 72 hr of culture as was TGF-beta present throughout 72 hr of culture. From 12 hr in culture to 24 hr, hepatocyte EGF binding sites dropped from about 230,000 to 85,000 per cell with no significant change in Kd, but with a loss in capacity for EGF-induced receptor down-regulation. Maximally inhibitory concentrations of TGF-beta did not compete with EGF for the EGF receptor, and a 4- to 24-hr exposure to TGF-beta did not alter subsequent EGF binding. Coincubation of hepatocytes with TGF-beta and EGF did not influence the 60% reduction in EGF binding sites produced by EGF alone. In addition, TGF-beta did not prevent EGF-induced autophosphorylation of the 170,000 dalton EGF receptor in membranes from whole liver. Our studies suggest that TGF-beta regulates hepatocyte growth independently of changes in EGF receptor number, ligand affinity, or postbinding autophosphorylation.     

Additive effect of oestradiol-17 beta and serum on synthesis of deoxyribonucleic acid in guinea-pig endometrial cells in culture.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.     

Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.

Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.     

Distinct effects of transforming growth factor-beta on EGF receptors and EGF-induced DNA synthesis in primary cultured rat hepatocytes

Previous studies showed that transforming growth factor-beta (TGF-beta, 1 ng/ml) strongly inhibited DNA synthesis induced by epidermal growth factor (EGF) in primary cultures of adult rat hepatocytes. This paper reports that TGF-beta (4 ng/ml) caused marked increase of EGF-binding to cultured rat hepatocytes. The binding increased biphasically with time to a maximum after treatment with TGF-beta for 12 h. Scatchard analysis showed that adult rat hepatocytes had a single class of non-cooperative binding sites with a Kd of 1.5 nM, that there were 1.4 X 10(5) binding sites/cell, and that TGF-beta increased the number of binding sites without changing the Kd value. The increase in EGF binding sites by TGF-beta was dose-dependent and the dose that elicited the maximum increase was about 10 times that which inhibited DNA synthesis stimulated by EGF. These findings suggest that the effect of TGF-beta in modulating the EGF receptor is not directly related to that in inhibiting DNA synthesis in adult rat hepatocytes.     

Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.     

Influence of epidermal growth factor (EGF) on ruffling activity, pinocytosis and proliferation of cultivated human glia cells

Normal human glia cells in culture were studied with respect to ruffling activity, macro-pinocytosis and cell proliferation under standard culture conditions with 10% serum in the medium, in serum-free medium and after addition of epidermal growth factor (EGF) or serum to previously serum-free medium. Pinocytotic uptake of droplets of medium occurred only in relation to well developed ruffling membranes. Omitting the serum from the medium led to a drastic reduction in thymidine incorporation. The cells became slender under these conditions, and soon after the change of medium their ruffling activity and pinocytosis were almost completely abolished. Following the change to a medium containing 2 ng EGF/ml a rapid reappearance of ruffling and pinocytosis was observed. DNA synthesis, however, was not demonstrated until after 20 h, showing that ruffling and pinocytosis occurred before DNA synthesis had started. Thus EGF may initially induce conformational changes of the plasma membrane, resulting in its internalization due to formation of endocytotic vacuoles. The observed relationship between occurrence of well developed ruffling membranes, macro-pinocytosis and cell multiplication indicates that one of the functions of growth-promoting factors may be stimulation of plasma membrane turnover.     

Resumption of cell cycle in BALB/c-3T3 fibroblasts arrested by polyamine depletion: relation with "competence" gene expression

Serum deprivation arrests BALB/c-3T3 fibroblasts (clone A31) in G0 phase, where resumption of the cell division cycle can be induced by addition of serum or of specific growth factors in a defined sequence: PDGF (inducer of a state of "competence," characterized by the expression of a family of genes including c-myc), epidermal growth factor EGF and IGF1 (Leof et al., 1982, 1983). When exponentially growing A31 cells are placed for greater than or equal to 2 days in a medium containing the alpha-difluoromethylornithine (alpha DFMO), an irreversible inhibitor of ornithine decarboxylase, they become arrested in G1 phase as a consequence of polyamine depletion (Medrano et al., 1983). In the alpha DFMO-arrested cells, addition of putrescine (60 microM) in a culture medium containing 6% fetal calf serum (FCS), but not in serum-free medium, is sufficient to induce G1 progression and entry into S phase (as determined by 3H-thymidine incorporation). The level of "competence" mRNAs is high in alpha DFMO-arrested cells. After addition of putrescine in FCS-containing medium, these mRNAs continue to be present for at least 3 h. A large proportion of alpha DFMO-arrested cells can be induced to progress to S phase by insulin (1 microM, acting via IGF1 receptor) plus putrescine in a serum-free medium (greater than or equal to 50% of FCS effect). In this case, the levels of "competence" mRNAs become low or undetectable within 3 h, EGF (10 nM) plus insulin had only slightly greater effect than insulin alone on the progression of alpha DFMO-arrested cells. When the alpha DFMO-arrested cells are subsequently incubated during 3 days in a low-serum-containing medium (0.25% FCS), they do not replenish their supply of polyamines, and then continue to express the c-myc gene. The recruitment of the polyamine-depleted, serum-deprived cells into the cell division cycle does not require PDGF and can be induced by addition of EGF and insulin plus putrescine. These data indicate that alpha DFMO arrests majority of the cells at a point situated beyond the PDGF- and EGF-dependent portion of G1 phase. During the subsequent serum deprivation, the alpha DFMO-arrested cells remain "competent" (PDGF-independent), probably as a consequence of their continued expression of c-myc (and possibly other PDGF-inducible genes).     

Effects of acidic fibroblast growth factor and epidermal growth factor on subconfluent fetal rat calvaria cell cultures: DNA synthesis and alkaline phosphatase activity

The effects of acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) were examined in subconfluent fetal rat calvaria cell cultures, in the presence of 2% serum. Maximal effect of aFGF and EGF on DNA synthesis measured by [³H]thymidine incorporation was observed after 18 h. aFGF stimulated DNA synthesis by 3.5-fold with an ED₅₀ of 0.75 ng/ml while a 2.3-fold EGF stimulation was recorded with an ED₅₀ of 0.067 ng/ml. 5-Bromo-2-deoxyuridine staining showed a higher stimulation of proliferation in the scattered cells than in the cell clusters. An 18 h aFGF or EGF treatment decreased alkaline phosphatase (ALP) activity by 40 and 23%, respectively, as compared with control cultures. This inhibition was more pronounced after 48 h in the presence of the effectors but no modification of the ALP electrophoretic mobility was observed. These data suggest that aFGF is a less potent mitogen than EGF and a higher inhibitor of ALP activity in fetal rat calvaria cell culture.     

Cell Cycle-Specific Requirement for Mevalonate, but Not for Cholesterol, for DNA Synthesis in Glial Primary Cultures

The requirement for the sterol biosynthetic pathway for the occurrence of DNA synthesis in glial cells and, in particular, the relative roles of cholesterol and of mevalonate have been studied. Primary cultures of developing glial cells were synchronized by reducing the content of fetal calf serum (FCS) in the culture medium from 10% to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the resulting quiescent state by the return of the cultures to 10% serum caused after 24 h a marked increase in DNA synthesis, and this increase was prevented by the simultaneous addition of mevinolin, a specific inhibitor of the sterol biosynthetic pathway at the 3-hydroxy-3-methylglutaryl coenzyme A reductase step, at the time of serum repletion. A dose-dependent reversal of the mevinolin inhibition of DNA synthesis occurred with simultaneous addition of mevalonate to the culture medium. The induction of DNA synthesis by serum repletion, its inhibition by mevinolin, and the reversal of the inhibition by mevalonate were unaffected by a 95% reduction in exogenous cholesterol produced by utilization of lipoprotein-poor serum (LPPS) rather than FCS. Similarly, return of quiescent cultures to 10% LPPS containing mevinolin and sufficient low-density lipoprotein (LDL) to raise the cholesterol concentration 80-fold failed to restore DNA synthesis. In addition, reversal of the mevinolin inhibition of DNA synthesis by mevalonate occurred despite the continuous presence of mevinolin if mevalonate was added as late as 12 h after serum repletion, but not if added after 16 h or more.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal regulation of DNA synthesis in primary cultures of adult rat hepatocytes : Action of glucagon

Primary cultures of adult rat hepatocytes, grown in modified minimal essential medium (Eagle's) containing 10% calf serum, could be induced into DNA replication by combinations of epidermal growth factor (EGF), insulin and glucagon. The three hormones acted synergistically, and cells began entering DNA synthesis 48 h after hormone addition. The ability of the hormones to stimulate DNA synthesis was enhanced by plating cells at high cell concentrations or by conditioned medium, and was diminished by daily medium change. The contribution of glucagon to DNA synthesis was replaced by cAMP plus 1-methyl, 3-isobutyl xanthine or by adrenergic agents. Evidence is presented which suggests that all three hormones are required on the first day of culture, and that EGF and insulin are also required after the first day. This appears to be a useful system for studies on the hormonal initiation of growth in quiescent cells.     

Inhibitory effect of a long-acting somatostatin analogue on EGF-stimulated cell proliferation in Capan-2 cells.

Numerous studies have reported diverse effects of gut-derived regulatory peptides on growth of the normal pancreas, pancreatic neoplasms induced experimentally in animals, and pancreatic cancer cell lines, but the results of these investigations are rather controversial. The stimulatory effect of epidermal growth factor (EGF) on cell proliferation of pancreatic cell lines is well established. Whether this action can be modulated by somatostatin is not clear. Furthermore, it is not certain whether another regulatory peptide, cholecystokinin (CCK), affects the proliferation of these cells. In the present study we investigated the presence of CCK-A and CCK-B, as well as somatostatin-2 (SSTR2) receptors by RT-PCR, and studied the actions of EGF, CCK and octreotide on DNA synthesis in the human pancreatic adenocarcinoma cell line Capan-2. Octreotide, a long-acting somatostatin analogue was used as somatostatin agonist. Cells were cultured in RPMI-1640 medium. They were incubated in serum free medium containing 0.2% BSA in the absence (control) or the presence of the peptides. [3H]-thymidine incorporation into DNA was measured after 48 h of incubation. By means of RT-PCR analysis we were able to demonstrate SSTR2 expression, but not CCK-A or CCK-B receptor mRNA in Capan-2 cells. DNA synthesis evaluated by [3H]-thymidine incorporation was found to be increased by 45.2 +/- 5.6% in response to EGF (10(-8) M) and decreased by 11.7 +/- 2.6% to octreotide (10(-8) M) compared to controls (P < 0.01). The increase in [3H]-thymidine incorporation was significantly lower when EGF treatment was combined with octreotide administration (10.1 +/- 2.5% over control). In the concentration range of 10(-11)-10(-8) M, CCK did not alter significantly the incorporation of [3H]-thymidine into DNA in Capan-2 cells. In conclusion, these data support a role for EGF as a growth factor for the human pancreatic cancer cell Capan-2. Somatostatin may play an important role in regulating cell proliferation in Capan-2 cells both under basal, and growth factor-stimulated conditions. Our results suggest, however, that CCK receptors are not expressed, and CCK does not affect cell proliferation in this transformed pancreatic cell line.