Butyrate blocks the accumulation of CDC2 mRNA in late G1 phase but inhibits both the early and late G1 progression in chemically transformed mouse fibroblasts BP-A31

Sodium butyrate (6 mM) blocks the resumption of the cell division cycle in serum-deprived chemically transformed Balb/c-3T3 mouse fibroblasts (BP-A31). The inhibition of G1 progression by sodium butyrate is not restricted to a specific mitogenic signaling pathway and is equally effective when tetradecanoyl phorbol acetate (TPA), insulin, or fetal calf serum (FCS) is used as inducer. The inhibitor acts in early as well as late G1 phase as indicated by experiments in which inhibitor was added and withdrawn at different times after restimulation of quiescent cells by FCS. At the gene expression level, sodium butyrate does not affect the inducibility of early cell cycle-related genes (c-myc, c-jun) while blocking the induction of cdc 2 mRNA, a late G1 marker. We conclude that sodium butyrate does not interfere with the growth factor signaling pathways regulating the (early) cell cycle-related gene expression. However, the presence of sodium butyrate early in G1 phase inhibits the cascade of events leading eventually to the expression of late G1-characteristic genes such as cdc2. The antimitogenic activity of sodium butyrate may be related to its interference with an (unknown) process involved in the "mitogenic" cascade.     

Sodium butyrate inhibits the phosphorylation of the retinoblastoma gene product in mouse fibroblasts by a transcription-dependent mechanism

In the chemically transformed mouse fibroblasts BP-A31, the retinoblastoma protein (pRb) is hypophosphorylated at quiescence and becomes hyperphosphorylated after approximately 6 h of serum stimulation. Phosphorylation of pRb was blocked if sodium butyrate was added together with serum or within 3 h afterwards. Actinomycin D added 3 h after serum stimulation did not prevent pRb phosphorylation, but it reversed the inhibitory effect of butyrate. These observations indicate that sodium butyrate acts by turning on the expression of gene(s) coding for proteins which prevent the accumulation of hyperphosphorylated pRb. Such butyrate-induced inhibitor(s) may interfere with the phosphorylation of pRb by cyclin-dependent kinases. Treatment of quiescent BP-A31 cells with serum in the presence of sodium butyrate has led to an increased cell content of the Waf1/CIP1 mRNA (coding for a cyclin-dependent kinase inhibitory protein) compared with serum alone, suggesting a possible role of p21Waf1/CIP1. In contrast, the mitogen activated protein kinase (enzyme which has been shown to phosphorylate pRb) was constitutively active in BP-A31 cells, and its activity was not significantly affected by a ≤ 3 h incubation with sodium butyrate. © 1996 Wiley-Liss, Inc.     

Mitogenic activity of phorbol esters and insulin-like growth factor 1 in chemically transformed mouse fibroblasts BP-A31: independent effects and differential sensitivity to inhibition by 3-isobutyl-1-methyl xanthine

Mitogenic effects of agents activating either the protein kinase C (PDGF; phorbol esters) or the insulin-like growth factor 1 (IGF1)-receptor pathway were studied in quiescent chemically transformed mouse fibroblasts (BP-A31), by evaluating the rate of [3H]thymidine incorporation. Each of these pathways alone was found to be sufficient to sustain progression through the entire cell division cycle. The mitogenic activity of phorbol 12-myristate 13-acetate (PMA) but not that of insulin was blocked by staurosporine (an inhibitor of protein kinase C), in support of the notion that protein kinase C activation was required for the PMA-induced cell cycle progression. The mitogenic effects of PMA were potentiated by cycloheximide pretreatment, and they were abolished by 3-isobutyl-1-methyl xanthine (IBMX; a cyclic nucleotide phosphodiesterase inhibitor). PDGF (known to activate the phospholipase C-protein kinase C pathway) also displayed mitogenic activity in the cycloheximide-pretreated BP-A31 cells, and its effects were prevented by IBMX. In contrast, the mitogenic effects of insulin (at concentrations where it activates the IGF1 receptor) or of IGF1 neither were notably influenced by cycloheximide pretreatment nor were inhibited by IBMX (in the presence of IBMX, the onset of S-phase was delayed by several hours). The expression of the c-fos gene was absent at quiescence; its induction by growth factors was not proportional to their mitogenic potency. Thus, c-fos expression was strongly induced by PMA but only weakly by insulin. IBMX was a powerful inducer of c-fos gene expression but caused a decrease in the level of c-myc mRNA.     

Fibroblast growth factor-dependent mitogenic signal transduction pathway in chemically transformed mouse fibroblasts is similar to but distinct from that initiated by phorbol esters.

Treatment of the quiescent, chemically transformed Balb/c mouse 3T3 cells (BP-A31) with fibroblast growth factor (FGF) leads to reinitiation of the cell division cycle in a large proportion of the cells. The characteristics of the mitogenic action of FGF closely resemble those of phorbol esters (activators of protein kinases type C) and differ from those of insulin (mediated by insulin-like growth factor 1 receptors). In particular, the effects of FGF as well as of phorbol-2-myristate-13-acetate (PMA), unlike the effects of insulin, are prevented by a low concentration (7.5 nM) of staurosporin (an efficient inhibitor of protein kinase C) as well as by 3-isobutyl-1-methyl xanthin (IBMX). Both FGF and PMA are good inducers of the accumulation of c-fos and c-jun mRNAs, whereas insulin has little effect. However, FGF was fully active (both as a mitogen and as inducer of c-fos mRNA accumulation) also in cells where the protein kinase C-mediated pathway had been downregulated by a long exposure to phorbol dibutyrate. We propose that the mitogenic effect of FGF does not require activation of protein kinase C, but that the subsequent events in the transduction pathways initiated by FGF and PMA, respectively, are (in part) coincident.     

Calcium requirements for mitogenic stimulation of 3T3-cells in low and high serum concentration

The calcium requirements during mitogenic stimulation of quiescent 3T3-cells in low or in high serum concentration was investigated. It was found that calcium played an equally important role for growth stimulation in low as in high serum concentration. Calcium was not required during the first 6 hours after mitogenic stimulation. However, calcium had to be present thereafter for the cells to initiate DNA-synthesis. The absence of calcium during the first six hours after onset of stimulation did not delay the initiation of DNA-synthesis.     

Cell cycle dependent regulation of cdc2 mRNA in mouse fibroblasts: Requirement of protein synthesis and of continued mitogenic stimulation

In the chemically transformed mouse fibroblasts (BP-A31) placed in a serum-free medium, the cdc2 mRNA content decreases in parallel with the cessation of [³H]thymidine incorporation. Extinction of the cdc2 gene expression is also observed in BP-A31 cells overexpressing the human c-myc oncogene. At quies-cence, the cdc2 gene expression can be reinduced with serum or with other mitogens such as insulin or 12-O-tetradecanoyl phorbol 13-acetate (TPA). The kinetics of induction is characterized by a lag period which differs according to the mitogen used and reflects the length of the G1 phase (4–6 h with insulin or serum, 9–12 h with TPA). The cdc2 mRNA accumulation is prevented when protein synthesis is blocked with cycloheximide, also if the drug is added at a time when the synthesis of cdc2 mRNA is already under way. Similarly, removal of the mitogen leads to a cessation of the cdc2 mRNA accumulation. These results suggest that the increased expression of the cdc2 gene is mediated by (a) short-lived, growth factor-regulated protein(s). © 1993 Wiley-Liss, Inc.     

The SPARC-related factor SMOC-2 promotes growth factor-induced cyclin D1 expression and DNA synthesis via integrin-linked kinase

Secreted modular calcium-binding protein-2 (SMOC-2) is a recently-identified SPARC-related protein of unknown function. In mRNA profiling experiments we, found that SMOC-2 expression was elevated in quiescent (G0) mouse fibroblasts and repressed after mitogenic stimulation with serum. The G0-specific expression of SMOC-2 was similar to that of platelet-derived growth factor-beta receptor (PDGFbetaR), a major mitogenic receptor. Therefore, we tested a possible role for SMOC-2 in growth factor-induced cell cycle progression. SMOC-2 overexpression augmented DNA synthesis induced by serum and fibroblast mitogens (including PDGF-BB and basic fibroblast growth factor). Conversely, SMOC-2 ablation by using small interfering RNA attenuated DNA synthesis in response to PDGF-BB and other growth factors. Mitogen-induced expression of cyclin D1 was attenuated in SMOC-2-ablated cells, and cyclin D1-overexpressing cells were resistant to inhibition of mitogenesis after SMOC-2 ablation. Therefore, cyclin D1 is limiting for G1 progression in SMOC-2-deficient cells. SMOC-2 ablation did not inhibit PDGF-induced PDGFbetaR autophosphorylation or PDGF-BB-dependent activation of mitogen-activated protein kinase and Akt kinases, suggesting that SMOC-2 is dispensable for growth factor receptor activation. However, integrin-linked kinase (ILK) activity was reduced in SMOC-2-ablated cells. Ectopic expression of hyperactive ILK corrected the defective mitogenic response of SMOC-2-deficient cells. Therefore, SMOC-2 contributes to cell cycle progression by maintaining ILK activity during G1. These results identify a novel role for SMOC-2 in cell cycle control.     

Hyperthermia can enhance the initiation of DNA synthesis stimulated by growth factors in Swiss mouse 3T3 cells

Confluent quiescent Swiss mouse 3T3 cells can be stimulated to initiate DNA synthesis and to divide by epidermal growth factor (EGF) and prostaglandin F2 alpha (PGF2 alpha), two mitogens of unrelated structure. Heat treatment at 46 degrees C for up to 20 min of confluent quiescent cells, which has no mitogenic effect, can enhance the stimulatory effect of suboptimal concentrations of EGF or PGF2 alpha on the initiation of DNA synthesis. Furthermore, insulin, which is not mitogenic in these cells, enhances the effect of these mitogens, but this effect is not further enhanced by heat treatment. Likewise the combination of EGF and PGF2 alpha is synergistic on DNA synthesis, and this effect is also not enhanced by the heat treatment. Incubation at 46 degrees C for longer than 20 min was inhibitory in all cases. These results suggest that heat treatment induces events which affect the regulation of the initiation of DNA synthesis in a manner depending on the duration of the heat treatment and the stimulation of the cells.     

Growth-mediated, density-dependent inhibition of endocytosis in cultured arterial smooth muscle cells

The effects of cell density and growth upon fluid phase endocytosis were investigated in quiescent and growing cultures of monkey arterial smooth muscle cells. Cells were maintained in a quiescent state of growth in 5% plasma-derived serum. Subsequent exposure of subconfluent cultures to the specific mitogens, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), or to whole blood serum, resulted in up to 4-fold increases in the rate of fluid endocytosis/cell. The changes began several hours after entry into G1 phase of the cell cycle and continued through S. The fraction of cells entering the growth cycle was variable (PDGF=FGF>EGF) and a close correlation existed between the rate of endocytosis and the fraction of [³H]thymidine-labelled cells (r = 0.929, p<0.01). At a range of cell densities, the rate of fluid endocytosis/cell was similar in sparse, confluent and post-confluent cultures of quiescent cells; in contrast, in growing cells there was density-dependent inhibition of endocytosis. Furthermore, when quiescent cells were in contact with each other and were then exposed to mitogens, the growth response was diminished and there was only a 25–50% increase in the rate of endocytosis, even in the presence of high concentrations of growth factors.These studies indicate that the influence of cell density upon fluid endocytosis in arterial smooth muscle cells is indirect in that it represents a secondary effect of decreased mitogenic response to specific growth factors.     

The mitogenic activity of pp60v-src, the oncogenic protein product of the src gene of avian sarcoma virus, is independent of external serum growth factors

The oncogenic pp60v-src product of ASV (avian sarcoma virus) is shown to be a potent endogenous mitogen, which, unlike mitogens such as PDGF (platelet derived growth factor), is able to stimulate host cell proliferation without the help of other growth factors. Thus, NRK rat cells, infected with a temperature-sensitive ASV mutant which produces an abnormally thermolabile pp60v-src, became proliferatively quiescent at a pp60v-src-inactivating 40 degrees C in medium containing either 0.2% calf serum or no serum at all. Adding PDGF stimulated the quiescent tsASV-NRK cells at 40 degrees C to initiate DNA replication in medium containing 0.2% serum, but not in serum-free medium. By contrast, activating internal pp60v-src by dropping the temperature to a permissive 36 degrees C stimulated these quiescent cells to transit G1, initiate DNA replication and to enter mitosis even in serum-free medium. Thus, relative to PDGF, endogenous pp60v-src behaves as a complete mitogen.     

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.     

Increased glutathione levels in quiescent, serum-stimulated NRK-49F cells are associated not with a response to growth factors but with nutrient repletion.

Treatment of quiescent cells with serum results concomitantly in an increase in cellular glutathione (GSH) content and growth stimulation. A possible association between the GSH increase and the growth response was examined by studying separately the effects of nutrients and growth factors on the levels of cellular GSH and proliferation of quiescent NRK-49F cells. The addition of fresh medium with 10% calf serum was found to result in both a twofold increase in cellular GSH and growth stimulation (DNA synthesis and cell proliferation). 10% calf serum alone, without fresh medium, stimulated cell growth but failed to cause a comparable increase in cellular GSH. The addition of fresh medium without 10% serum, and of 0.5 mM cysteine and glutamate, resulted in both instances in a marked increase in cellular GSH, but failed to stimulate cell growth. EGF, in contrast, induced a complete mitogenic response but did not increase cellular GSH. Finally, pretreatment with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of GSH synthesis, decreased cellular GSH and inhibited EGF-induced DNA synthesis, but these two responses do not, in their dose dependency, correlate. The results obtained thus show that the increase in cellular GSH that occurs in quiescent, serum-stimulated NRK-49F cells is a result of nutrient repletion rather than mitogenic stimulation, and increased GSH levels do not necessarily precede DNA synthesis and mitosis.     

Studies on the nature of protease-induced growth stimulation in normal and transformed BHK cells.

In the presence of growth-limiting serum concentrations trypsin displays mitogenic activity on actively-growing but not quiescent BHK cells. These results suggest that BHK cells arrested in G1 (G0) are not sensitive to protease-induced growth stimulation. Previous work strongly suggested that the trypsin active-site is not directly involved in its mitogenic activity on BHK cells. Additional studies on denatured trypsin fragments further indicate that the molecular conformation and size of native trypsin may not be absolutely required for mitogenic activity. Cellular multiplication induced by the addition of fresh serum to quiescent BHK cultures is not inhibited by high concentrations of soybean trypsin inhibitor. Similar to our previous findings with trypsin, it has been further observed that plasmin is not sufficient to initiate the growth of BHK cells in soft agar. Trypsin also fails to enhance the growth of a thermosensitive polyoma-transformed BHK line in soft agar at the restrictive temperature. Finally, the growth of transformed BHK cells in soft agar does not display a requirement for plasminogen and is not inhibited by soybean trypsin inhibitor. These studies argue against the involvement of plasmin or other exogenous trypsin-like enzymes in the growth and transformation of BHK cells.     

Stimulation of bumetanide-sensitive Na+/K+/Cl- cotransport by different mitogens in synchronized human skin fibroblasts is essential for cell proliferation

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl- cotransport in the mitogenic signal of human skin fibroblast proliferation. The Na+/K+/Cl- cotransport was dramatically stimulated by either fetal calf serum, or by recombinant growth factors, added to quiescent G0/G1 human skin fibroblasts. The following mitogens, FGF, PDGF, alpha-thrombin, insulin-like growth factor-1, transforming growth factor-alpha, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, all stimulated the Na+/K+/Cl- cotransport. In addition, all the above mitogens induced DNA synthesis in the synchronized human fibroblasts. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal, the effect of two specific inhibitors of the cotransport, furosemide and bumetanide, was tested on cell proliferation induced by the above recombinant growth factors. Bumetanide and furosemide inhibited synchronized cell proliferation as was measured by (a) cell exit from the G0/G1 phase measured by the use of flow cytometry, (b) cell entering the S-phase, determined by DNA synthesis, and (c) cell growth, measured by counting the cells. The inhibition by furosemide and bumetanide was reversible, removal of these compounds, completely released the cells from the block of DNA synthesis. In addition, the two drugs inhibited DNA synthesis only when added within the first 2-6 h of cell release. These results indicate that the effect of these drugs is specific, and is not due to an indirect toxic effect. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport plays a major role in the mitogenic signaling pathway of the human fibroblasts.     

A partially purified serum fraction synergistically enhances the mitogenic activity of epidermal growth factor and insulin in quiescent cultures of 3T3 cells.

Epidermal growth factor and insulin need a low concentration of serum to effectively stimulate quiescent 3T3 cells into DNA synthesis. We partially purify a polypeptide component of serum which has no activity by itself but which acts synergistically with epidermal growth factor and insulin to stimulate cultures of 3T3 cells into DNA synthesis as effectively as whole serum. The active fraction is separated from serum by gel chromatography on Sephadex G-100, under acid dissociating conditions, and chromatographs with a molecular weight of 18,000 daltons.We suggest as a general technique, the use of pure growth factors to assay for growth promoting fractions from serum or other sources. Fractions which are not mitogenic by themselves can be detected when assayed together with their complementary pure factors.     

Reinitiation of DNA synthesis in quiescent mouse keratinocytes; Regulation by polypeptide hormones,cholera toxin,dexamethasone, and retinoic acid

Summary Cloned mouse keratinocytes (MK-1 cells) display density-dependent growth arrest when reaching confluency in a serum-free medium with a calcium concentration <0.1 mM, supplemented only with insulin and transferrin. In this quiescent state, greater than 95% of the cell population is in the G_0/1 phase of the cell cycle. Treatment of quiescent MK-1 cells with 1 to 10 ng/ml epidermal growth factor (EGF) resulted in a sharp burst of DNA synthetic activity. Both insulin and cholera toxin potentiated the mitogenic effect of EGF, but neither agent was necessary or sufficient to induce thymidine incorporation into DNA. Dexamethasone abolished the effect of insulin, but not the mitogenic effect of EGF alone. In contrast, retinoic acid (RA) did not possess any mitogenic effect for quiescent MK-1 cells, nor did it modulate the actions of EGF or dexamethasone. A number of commercially available crude extracts of bovine brain and pituitary were also capable of initiating DNA synthesis in resting MK-1 cells. Finally, transforming growth factor type beta (TGFβ) proved to be a potent inhibitor of the mitogen-induced DNA synthesis in MK-1 cells (IC₅₀∶10pM). This defined culture system is eminently suited to study the regulation of DNA synthesis of epidermal cells. In addition, it can be used as a sensitive bioassay for the detection of epidermal mitogens, as well as inhibitors of DNA synthesis such as TGFβ. Supported by PHS Award CA-41556 from the National Cancer Institute, Bethesda, MD.     

Stimulation of bumetanide-sensitive K+ transport in Swiss 3T3 fibroblasts by serum and mitogenic hormones

Rapidly growing Swiss 3T3 fibroblasts possess a bumetanide-sensitive K+ transport system that is dependent on both Na+ and Cl- ions; a smaller bumetanide-insensitive component of K+ transport is also present. In cells brought to the quiescent state by 8-11 days of incubation without a medium change, the bumetanide-sensitive rate of transport was reduced by 63%; the bumetanide-insensitive rate did not change. Removal of dialyzed fetal calf serum from the uptake medium resulted in a substantial reduction in bumetanide-sensitive uptake in both rapidly growing cells (33% reduction) and quiescent cells (68% reduction) but had no effect on bumetanide-insensitive uptake. Insulin was almost as effective as dialyzed fetal calf serum in stimulating bumetanide-sensitive uptake; insulin was maximally stimulatory at 2.5 micrograms/ml. The combination of insulin, epidermal growth factor, and arginine-vasopressin was maximally effective in stimulating both bumetanide-sensitive K+ uptake and 3H-thymidine incorporation in quiescent cells; bumetanide, however, did not interfere with the hormonal stimulation of DNA synthesis. Thus, the bumetanide-sensitive K+ transport system is not necessary for such stimulation to occur. Furthermore, concentrations of hormones which stimulated significant levels of DNA synthesis produced no elevation in the intracellular concentration of K+. We conclude that the bumetanide-sensitive pathway of K+ transport is modulated by serum and by mitogenic hormones, but does not play a role in the stimulation of DNA synthesis by these factors.     

Altered cellular responses to serum mitogens, including platelet-derived growth factor, in cultured smooth muscle cells derived from arteries of patients with moyamoya disease

Progressive stenosis or occlusion of bilateral internal carotid arteries by fibrocellular intimal thickening results in cerebral ischemia in moyamoya disease. The etiology is unknown. We examined cultured arterial smooth muscle cells (SMC) from scalp arteries of five patients with moyamoya disease. In this study we investigated the responsiveness of the cells in culture to serum mitogens including platelet-derived growth factor (PDGF), a major mitogen of SMC, and compared the response to that of cells derived from age-matched control patients. SMC from patients with moyamoya disease proliferated less rapidly in a medium with 15% serum than did control SMC and responded poorly to the addition of PDGF to 5% serum. PDGF alone did not stimulate SMC in a quiescent state to initiate DNA synthesis in moyamoya disease, without serum factors other than bovine serum albumin, though it significantly stimulated the controls. Simultaneous additions of epidermal growth factor, insulin-like growth factor-I, and PDGF stimulated initiation of DNA synthesis in cells from moyamoya disease, but not as much as PDGF alone did in the controls. Although direct correlations with the pathogenesis of the disease remain to be clarified, the results indicate altered interrelations between serum factors and the cellular responses in vessels of moyamoya disease.     

Insulin induces a selective heterologous desensitization of the mitogenic response of Swiss 3T3 cells to vasopressin

Prior incubation of confluent, quiescent cultures of Swiss 3T3 cells with insulin leads to a selective loss of mitogenic stimulation on re-addition of the combination of vasopressin and insulin in serum-free medium. The desensitization is specific for the action of vasopressin as insulin is fully active in the refractory cells when added in combination with other mitogens, whereas vasopressin is not. A prolonged treatment with insulin is required for induction of the refractoriness, half-maximal loss of response occurs after about 7 h and desensitization is complete after 12 h treatment. The refractory cells recover their response to vasopressin after more than 24 h incubation in the absence of insulin. A rapid response of the cells to vasopressin, inhibition of 125I-epidermal growth factor (125I-EGF) binding, is also desensitized by insulin. Desensitization is induced by insulin-like growth factor I (IGF-I), and partially by desoctapeptide insulin, but not by insulin B chain. Although the characteristics of insulin-induced desensitization are very similar to those of the homologous desensitization induced by vasopressin treatment, insulin does not bind to vasopressin antiserum or the [3H]vasopressin receptors of Swiss 3T3 cells. Insulin treatment also does not lead to any down-regulation of [3H]vasopressin receptors, and the refractoriness of the cells must therefore lie at a post-receptor step. Both insulin- and vasopressin-induced refractoriness to the mitogenic action of vasopressin can be blocked by a low level of cycloheximide. Both these agents therefore seem to induce the synthesis of specific protein(s) which selectively inhibit the mitogenic response of the cell to vasopressin.