Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Prolactin-dependent mitogenesis in Nb 2 node lymphoma cells: effects of immunosuppressive cyclopeptides

Prolactin (PRL)-stimulated ornithine decarboxylase (ODC) activity and subsequent proliferation are inhibited by the cyclopeptides cyclosporine (CsA) and didemnin B (DB) in Nb 2 node lymphoma cells. Similar concentrations of these agents also inhibit 125I-PRL binding, suggesting that their inhibitory effects on these PRL-dependent physiologic responses are mediated at least in part at the level of PRL receptor interactions. The phorbol ester TPA stimulated ODC activity and [3H]thymidine incorporation to 54% and 31% that of a near-optimal mitogenic concentration of PRL (10 ng/ml), suggesting that mitogenesis in these cells is coupled to some degree to the activation of protein kinase C (PKC). The calcium ionophore A23187 increased ODC activity only slightly and actually decreased [3H]thymidine incorporation to a value below the "cells only" controls. The addition of TPA plus A23187 did not further enhance the effects of TPA to elevate ODC activity and [3H]thymidine incorporation. However, A23187 significantly elevated PRL-stimulated ODC activity with a subsequent inhibition of [3H]thymidine incorporation, suggesting a block of entry into S phase. Both cyclopeptides decreased the elevation of ODC activity in G1 phase of cell cycle in response to PRL, suggestive of a site of action for these agents in early G1, a conclusion compatible with their ability to inhibit PRL binding to these cells. Addition of CsA or DB 2 hr after PRL had no effect on PRL-stimulated ODC activity detectable at 6 hr, but addition of either as late as 6 hr still affected the extent of mitogenesis. This is in line with the requirement for PRL to be present in the culture medium for a minimum of 3 to 6 hr to invoke a maximal effect on mitogenesis. Addition of either cyclopeptide after the cells were in S phase had no effect on the extent of [3H]thymidine incorporation. An inhibitor of the cyclooxygenase pathway (indomethacin) enhanced both PRL-stimulated ODC activity and proliferation, whereas inhibition of the lipoxygenase pathway by NDGA attenuated only proliferation, suggesting that in Nb 2 cells, products of the lipoxygenase pathway may contribute to the mechanism of PRL-stimulated mitogenesis. Because Nb 2 lymphoma cells were derived from estrogenized rats, estrogen was tested as a mitogen. By itself it was not mitogenic, but in conjunction with PRL, estradiol-17 beta elevated the ODC response and inhibited proliferation. Inhibitors of PKC known to have minimal effects on RNA synthesis, quercetin and gossypol, totally inhibited both the elevations of ODC activity and [3H]thymidine incorporation in response to PRL in Nb 2 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prolactin and growth hormone synthesis and thymidine incorporation in dissociated rat pituitary tumor cells

The effect of in vivo diethylstilbestrol (DES) treatment on the MtT/W15 transplantable pituitary tumor was examined in dissociated pituitary cells by measuring the rate of incorporation of [3H]thymidine into DNA and the synthesis of prolactin (PRL) and growth hormone (GH) as assessed by the rate of incorporation of [3H]leucine. MtT/W15 transplantable pituitary tumors from rats treated for 3 weeks with DES showed significant reduction in the extent of [3H]thymidine incorporation compared with tumor cells from untreated rats (2231 +/- 182 vs 172 +/- 17 dpm/10(5) cells; n = 3). In addition, tumor cells from DES-treated rats showed a significant increase in GH synthesis compared with tumor cells from untreated rats. In contrast to these findings, dissociated pituitary cells from non-tumor-bearing rats given 10 mg DES in Silastic tubing for 3 weeks showed a three-fold increase in PRL synthesis compared to cells from untreated control rats (29.3 +/- 1.5 vs 10.0 +/- 0.9% of total radioactivity in gel; n = 3. There was also a four-fold increase in the rate of [3H]thymidine incorporation after DES-treatment in non-tumor-bearing rats (695 +/- 114 vs 178 +/- 13.9 dpm/10(5) cells; n = 3). These results indicate that DES inhibits MtT/W15 pituitary tumor cell proliferation, while stimulating synthesis of GH.     

Enrichment of the poly (A) sequence and lack of enhancement of total RNA synthesis in cultured Xenopus hepatocytes by estradiol-17 beta

The effect of estradiol-17 beta on RNA synthesis and the amounts of total RNA and polyadenylic acid were determined in primary cultures of Xenopus laevis liver parenchymal cells. Results showed that estradiol did not alter the RNA content significantly; control cells contained 11.9 +/- 0.34 micrograms and estradiol-treated cells 12.4 +/- 0.17 micrograms per 10(6) cells on day 2 of estradiol treatment, and 22.0 +/- 0.61 micrograms and 24.0 +/- 1.09 micrograms on day 5. Hybridization with [3H]poly(U) revealed that estradiol increased the poly(A) content about 1.2-fold more than in the controls on day 2 and 1.6-fold on day 5 of estradiol treatment. The actual rate of RNA synthesis was estimated from analyses of the kinetics of [3H]adenosine incorporation into the ATP pool and into RNA. The initial rate of incorporation of ATP into RNA on day 5 of estradiol treatment was 29.38 pmol/min/10(6) cells and the rate of the controls of 29.35. Subsequent accumulation kinetics of [3H]adenosine into RNA showed no difference between estradiol and the control cells. Thus, estradiol did not alter the rate of total RNA synthesis and the total RNA content significantly, but it did increase the poly(A) content.     

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.     

13-cis retinoic acid and all-trans retinoic acid in the regulation of the proliferation and survival of human breast cancer cell line MCF-7.

Retinoids are a group of compounds which inhibit cell proliferation and induce cellular differentiation. The aim of this study was to compare the antiproliferative activity of various concentrations of 13-cis retinoic acid (isotretinoin) and all-trans retinoic acid (tretinoin) in a culture of the estrogen-sensitive human breast cancer cell line MCF-7. Evaluation was based on [3H]thymidine incorporation into the cancer cells and through immunocytochemical analysis of cell cycle-associated PCNA and Ki-67 protein expression. Both retinoids inhibited [3H]thymidine incorporation into the cancer cells most effectively at a concentration of 3x10(-3) M. Two basic substances used for line MCF-7 culture experiments, one stimulating - estradiol - and the other inhibiting - tamoxifen - were applied. Estradiol added to a culture containing decreasing concentrations of isotretinoin (from 3x10(-3) to 3x10(-8) M) caused a statistically significant reduction in the percentage of [3H]thymidine incorporation into the cancer cell line MCF-7, compared to the 17 beta estradiol group (189.25%+/-62.64, control=100%, p<0.05). In the group of decreasing tretinoin concentrations, statistically significant differences were found only at 3x10(-3), 3x10(-4) and 3x10(-8) M. Following culture supplementation with tamoxifen (1 microM), statistically significant differences were observed only at the highest concentrations of both retinoids (3x10(-3) and 3x10(-4) M). The evaluation of breast carcinoma cells with a positive immunocytochemical reaction to PCNA and Ki-67 has revealed that isotretinoin reduces their percentage in the most determined and statistically significant way (38.00%+/-2.58 and 39.25%+/-3.09), compared to the control group (86.50%+/-9.20 and 100%+/-3.87, p<0.001 and p<0.0001) and to the estradiol group (87.00%+/-6.79 and 86.10%+/-7.0, p<0.001). Apart from their blocking effect on the cell cycle, retinoids also induce the apoptotic pathway.     

Oxytocin stimulates proliferation of human osteoblast-like cells

Oxytocin receptors have recently been demonstrated in human osteoblast-like (hOB) cells. In this study, oxytocin 100-1000 pmol/l increased cell proliferation of primary cultures of hOB cells, measured by [3H]thymidine incorporation, (P<0.01). In human osteosarcoma cell-line (SaOS-2), oxytocin 100 pmol/l increased cell proliferation (measured by [3H]thymidine incorporation and a commercially available kit) and protein synthesis ([3H]proline incorporation) (P<0.05). The increase in cell proliferation was abolished when SaOS-2 cells were incubated with an oxytocin antagonist and oxytocin. Oxytocin 100 pmol/l decreased interleukin-6 (IL-6) production of the hOB cells (23.4+/-1.96 versus 33.4+/-2.65 pg/well; P<0.001). These findings indicate that oxytocin may affect bone metabolism in humans.     

Assays for in vitro monitoring of proliferation of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cells

Vascular and airway remodeling, which are characterized by airway smooth muscle (ASM) and pulmonary arterial vascular smooth muscle (VSM) proliferation, contribute to the pathology of asthma, pulmonary hypertension, restenosis and atherosclerosis. To evaluate the proliferation of VSM and ASM cells in response to mitogens, we perform a [3H]thymidine incorporation assay. The proliferation protocol takes approximately 48 h and includes stimulating cells synchronized in G0/G1 phase of the cell cycle with agonists, labeling cells with [3H]thymidine and examining levels of [3H]thymidine incorporation by scintillation counting. Although using radiolabeled [3H]thymidine incorporation is a limitation, the greatest benefit of the assay is providing reliable and statistically significant data.     

Synergism between prolactin and ovarian hormones of DNA synthesis in rats mammary gland.

Autoradiography with [30H] thymedine has been used to measure the rate of DNA synthesis in the mammary epithelium of hypophysectomized-ovariectomized rats under the influence of estradiol, progesterone, and prolactin. Controls and animals treated with estradiol did not increase [3-H] thymidine incorporation, while progesterone alone had a slight stimulatory effect. Prolactin alone stimulated some [3-H] thymidine uptake in ductal and alveolar epithelium, but when combined with either estradiol or progesterone synergistic effects were observed. Estradiol with prolactin stimulated incorporation primarily in the ductal epithelium, whereas progesterone with prolactin stimulated both ductal and alveolar epithelium.     

Inhibition by colchicine of the mitogenic stimulation of lymphocytes prior to the S phase

Colchicine, vinblastine, and vincristine inhibit the mitogenic stimulation of lymphocytes by concanavalin A as measured by the incorporation of [3H]thymidine and the appearance of blast cells. The inhibitory effect of colchicine could not be accounted for by diminution in cell viability or by metaphase arrest of mitosis in the stimulated cells. Moreover, the inhibition of [3H]thymidine incorporation was not due to blockage of thymidine transport or inhibition of DNA synthesis inasmuch as addition of colchicine had no effect on cells in the S phase of the cell cycle. The time of inhibition was correlated with the kinetics of cellular commitment to lectin activation and the kinetic data indicated that colchicine blocks stimulation early in the sequence of events following addition of the mitogen. These findings support the hypothesis that cytoplasmic microtubular function plays a role in the commitment of resting cells to undergo mitotic division.     

Activation of mitogen-activated protein kinase by oxidized low-density lipoprotein in canine cultured vascular smooth muscle cells

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase inhibitor genistein for 1h, substantially reduced the [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca(2+) by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca(2+) for these responses. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.     

Repair of daughter strand gaps in nascent DNA from mouse epidermal cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

Alkaline sucrose gradient analysis of [methyl-3H]thymidine-pulse-labeled DNA was used to study the effect of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide I), a potent mutagen and carcinogen, and (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide II), a weaker mutagen and carcinogen, on the size of newly synthesized DNA in primary cultures of mouse epidermal cells. Both isomers caused a dose-dependent decrease in the size of newly synthesized DNA and in the rate of [methyl-3H]thymidine incorporation into DNA. When the pulse time was increased in the treated cells so that the amount of [methyl-3H]thymidine incorporation was equal to the control, newly synthesized DNA from exposed cells was still considerably smaller than DNA from control cells. The low molecular weight of the nascent DNA from treated cells was consistent with, but not indicative of, the presence of gaps in the nascent DNA from the treated cells. Evidence of gapped DNA synthesis was obtained by treatment of extracted DNA with a single-strand specific endonuclease from Neurospora crassa. The endonuclease treatment did not significantly alter the profile of [methyl-3H]thymidine prelabeled DNA from benzo[a]pyrene-diol epoxide-treated cultures but did introduce double-stand breaks in pulse-labeled DNA from treated cultures. The numbers of [14C]benzo[a]pyrene-diol epoxide I or [3H]benzo[a]pyrenediol epoxide II-DNA-bound adducts and daughter strand gaps were compared at several dose levels. Treatment with either isomer yielded one gap in the nascent DNA/DNA-bound adduct. Pulse-chase experiments showed that gaps in the nascent DNA were closed with time.     

Specific modifications of hepatoma cell-surface glycoproteins with enzymes : Effects on in vitro growth as investigated by the use of lectins

The effects of enzymic treatment on the interactions between Zajdela's tumor cells and various lectins. Concanavalin A (ConA); Wheat Germ Agglutinin (WGA); Robinia lectin; have been studied. (1) The number of lectin-binding sites and the affinity constants were investigated. (2) The effects of the lectins on cell growth and [3H]thymidine incorporation were studied on untreated and enzyme-treated cells. It was observed that treatment of tumor cells with neuraminidase resulted in a change in the binding characteristics of each lectin. However, additional treatment of the cells with galactose oxidase had no further effect on lectin binding. ConA and Robinia lectin induced a decrease of the untreated tumor cell growth and a stimulation of the [3H]thymidine incorporation. This paradoxal result may be explained as a consequence of the stimulation of the [3H]thymidine uptake observed in the presence of lectins. The enzymatic treatments themselves did not change the cell growth although they did induce a change in the effect of ConA and Robinia lectin on cell growth and [3H]thymidine incorporation. As a result of neuraminidase treatment, the effects of ConA were totally suppressed but those of Robinia lectin only partially. Although WGA interacted with untreated and enzyme-treated cell surfaces, it had no effect on tumor cell growth nor [3H]thymidine incorporation. The results are discussed in terms of lectin transport.     

Double-stranded RNA regulation of DNA synthesis in fibroblasts.

The double-stranded RNA molecule polyinosinic-polycytidylic acid (poly IC) has been found in some studies to have a mitogenic effect on fibroblast proliferation while other studies found poly IC to have an inhibitory effect on proliferation. In this study, we investigated whether a stabilized form of poly IC complexed with poly-L-lysine and carboxymethylcellulose (poly ICLC) had a bidirectional effect on DNA synthesis in fibroblasts from four different cell lines and determined factors that potentially influence this bidirectional effect. In medium containing fetal bovine serum, poly ICLC slightly increased the levels of [3H]thymidine incorporation in growing fibroblasts in three of the four fibroblast cell lines tested, while poly ICLC increased [3H]thymidine incorporation in confluent, quiescent fibroblasts in two of four cell lines. Poly ICLC did not induce DNA synthesis in subconfluent, quiescent or in confluent, quiescent fibroblasts under serum-free conditions. Poly ICLC significantly suppressed serum-induced [3H]thymidine incorporation by quiescent fibroblasts in all cell lines. We conclude that the stimulatory and inhibitory effects of poly ICLC on DNA synthesis are influenced by both the cell line and the presence of serum components in the culture medium but not by population density.     

Human C-apolipoproteins promote hydrolysis of dimyristoyl phosphatidylcholine by snake venom phospholipase A2

Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10^?5 M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [³H]thymidine incorporation. Proliferation is completely restored by 5 × 10^?5 M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 × 10^?5 M each, fail to re-establish proliferation but produce an isotropic dilution of [³H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (<0.001 nmol.min^?1.10^?6 cells).     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Effects of bradykinin on DNA synthesis in resting NIL8 hamster cells and human fibroblasts

Bradykinin stimulates [3H]thymidine incorporation and DNA synthesis in resting, serum-deprived NIL8 hamster cells. The ED50 for this stimulation is 4.52 +/- 2.91 nM. Other kinin peptides including lys-bradykinin (kallidin) and met-lys-bradykinin also stimulate [3H]thymidine incorporation in the NIL8 cells, whereas desarg9-bradykinin is without effect, suggesting action of the kinin peptides through type B2 receptors. Bradykinin also stimulates DNA synthesis in IMR-90 human fibroblasts; however, this effect is observed only in the presence of indomethacin, which blocks prostaglandin synthesis. These results suggest that prostaglandins act as negative modulators of the growth-stimulatory effects of bradykinin in the fibroblasts. This conclusion is supported by the observation that exogenously added PGE1, PGE2, PGA1, PGA2, PGB1, and PGB2 strongly inhibit [3H]thymidine incorporation in the human fibroblasts. The direct effect of bradykinin observed in the NIL8 cells may be attributable to the relative resistance of these cells to growth inhibition by prostaglandins.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

Insulin-like growth factor II increases cytoplasmic free calcium in competent Balb/c 3T3 cells treated with epidermal growth factor

To determine the role of calcium in the action of insulin-like growth factor II (IGF-II), we have examined the effect of multiplication stimulating activity, the rat IGF-II, on cytoplasmic-free calcium concentration, [Ca2+]c, in aequorin-loaded Balb/c 3T3 cells. IGF-II does not cause any change in [Ca2+]c in quiescent cells. By contrast, IGF-II induces changes in [Ca2+]c in platelet-derived growth factor(PDGF) - pretreated competent cells: when competent cells are incubated with epidermal growth factor (EGF) for 10 min, subsequent IGF-II induces an immediate increase in [Ca2+]c. Without EGF treatment, IGF-II does not cause any increase in [Ca2+]c. The priming action of EGF is time dependent, requiring approximately 10 min for the maximum effect. The IGF-II-mediated increase in [Ca2+]c is totally dependent on extracellular calcium and is blocked by lanthanum. When DNA synthesis in PDGF-treated competent cells is assessed by measuring [3H]thymidine incorporation, IGF-II by itself has only a small effect. Likewise, a brief treatment with EGF results in only a small increase in [3H]thymidine incorporation. By contrast, in competent cells briefly treated with EGF, IGF-II causes a marked stimulation of [3H]thymidine incorporation. These results indicate that IGF-II increases [Ca2+]c in competent Balb/c 3T3 cells treated with EGF by stimulating calcium influx and that IGF-II-stimulated calcium influx may be related causally to its action on cell proliferation.     

Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.