Effects of reproductive activity and sex hormones on apoptosis in the epigonal organ of the skate (Leucoraja erinacea)

In elasmobranchs, a unique association exists between an immune tissue, the epigonal organ, and the gonads. The intimate morphological relationship between these tissues suggests functional interactions. In this study, we used apoptosis to assess differences between epigonal tissues of reproductively active (RA) and non-reproductively active (NRA) skates (Leucoraja erinacea). Plasma steroid levels were significantly higher in RA than in NRA animals, and TUNEL analysis showed that epigonal tissue of RA skates had greater DNA fragmentation than NRA skates. Addition of steroids to epigonal leukocytes in vitro demonstrated that progesterone, testosterone, and dexamethasone, but not estrogen, induced apoptosis of epigonal leukocytes as evidenced by DNA laddering and caspase-3 antibody labeling. This study supports recent evidence that cellular homeostasis of epigonal lymphomyeloid tissue may be influenced by gonadal activity and reproductive steroids in a representative of the most basal gnathastome group.     

Inhibition of Replication in Functional Mouse Adrenal Tumor Cells by Adrenocorticotropic Hormone Mediated by Adenosine 3′:5′-Cyclic Monophosphate

Adrenocorticotropic hormone (ACTH) inhibited replication in functional adrenal tumor cells with a concomitant stimulation of steroidogenesis and a characteristic change of morphology from a flattened to a spherical type. [(3)H]Thymidine incorporation into DNA was inhibited by about 50% 6 hours after ACTH treatment. Both cyclic AMP and dibutyryl cyclic AMP inhibited [(3)H]thymidine incorporation and caused the characteristic morphological change noted with ACTH. The extent of stimulation of steroidogenesis and the amount of inhibition of [(3)H]thymidine incorporation in response to various doses of ACTH were closely related and both were in parallel with the concentration of cyclic AMP in the cells. Cyclic GMP and cyclic IMP did not inhibit [(3)H]thymidine incorporation significantly, and did not change the morphology of the cells. AMP inhibited [(3)H]thymidine incorporation into DNA and caused the characteristic morphological change. However, AMP did not increase the cyclic AMP content of the cells. CMP, GMP, and UMP showed a significant inhibition of [(3)H]thymidine incorporation into DNA, but the extent of the inhibition was much less than that with AMP. These nucleotides did not change the morphology of the cells.     

周期素激酶抑制剂p27在肿瘤坏死因子-α诱导系膜细胞增生中的作用

目的 研究周期素激酶抑制剂p27在肿瘤坏死因子－α（TNF－α）诱导系膜细胞（MC）增生中的作用。方法 采用蛋白印迹（Western杂交）方法测定MC裂解液p27蛋白水平。[^3H]胸腺嘧啶核苷（[^3H]TdR）测定MC增生的情况,观察p27反义寡核苷酸（ODN）转染对TNF-α刺激MC中的p27水平及其对增生程度的影响。结果 TNF－α（200000U/L）可使无血清培养24h的MC中的p27水平降低（P＜0.01）,同时使[^3H]TdR掺入增加（P＜0.01）;p27反义ODN转染可降低TNF－α刺激24h的MC中的p27水平（P＜0.01）,同时可使[^3H]TdR掺入增加更为明显（P＜0.05）。结论 p27水平降低可能在TNF－α诱导MC增生中起重要作用。     

Regulation of pancreatic islet cell replication: an inquiry into the controversy regarding the effects of steroid hormones

The controversial issue of the effects of prednisolone and 17 beta-estradiol on replication of fetal rat pancreatic islets in culture was studied using 32P and [3H]thymidine as probes for studying DNA synthesis. DNA synthesis was not affected by the steroid hormones, as was evident from the rate of incorporation of 32P into total DNA. Decreased incorporation of [3H]thymidine into DNA found in islets treated with either of these steroids seemed to reflect an inhibitory effect of these hormones on thymidine kinase, leading to decreased phosphorylation of labeled thymidine. In addition, the hormones stimulated the activity of thymidylate synthetase, thus enhancing the endogenous synthesis of thymidine and thereby diluting the specific activity of the [3H]thymidine added to the cultured islets. Further support for a lack of inhibition of growth of islet cells treated with steroid hormones was provided by the observation that prednisolone increased uridine kinase activity and RNA biosynthesis, both of which may participate in the growth of cells preceding mitosis and (the latter) in protein hormone biosynthesis.     

Blastic Transformation of Mouse Spleen Lymphocytes by a Water-Soluble Mitogen Extracted from Nocardia

A water-soluble extract of Nocardia markedly increased in vitro [(3)H]thymidine incorporation by mouse spleen lymphocytes. The blastogenic activity of the extract and lipopolysaccharide was studied comparatively on various mouse lymphocyte subpopulations. The data obtained by [(3)H]thymidine incorporation and by electron microscopy have demonstrated that this preparation stimulates selectively mouse bone-marrow-derived cortisone-sensitive lymphocytes. This stimulation is related neither to a natural infection of mice with Nocardia organisms nor to the presence in Nocardia water-soluble mitogen of a lipopolysaccharide contaminant or of a lipopolysaccharide-like material.     

DNA Synthesis in Mixed Cultures of Rat Leukocytes and Allogeneic Dissociated Skin Cells

A proliferative response (increased DNA synthesis measured by [(3)H]thymidine incorporation; maximum at the fifth day) occurs in mixed cultures of rat peripheral leukocytes and allogeneic dissociated skin cells. This response is allospecific and is greater than that occurring in mixed cultures of corresponding allogeneic leukocytes. This mitogenic property of dissociated skin cells is not shared with other nonlymphoid cell types, for example kidney cells. Cells from four isogenic rat strains have been studied; proliferative responses occurred in the mixed cell cultures only when cell donors differed by alleles at the Ag-B histocompatibility locus.     

Molecular mechanism of progesterone-induced antiproliferation in rat aortic smooth muscle cells

Previously we demonstrated that progesterone at physiologic levels dose dependently inhibited cell proliferation in cultured rat aortic smooth muscle cells (RASMCs). However, the molecular mechanism underlying of progesterone-induced antiproliferation was not clear. Here we demonstrated that progesterone induced a reduction of the [(3)H]thymidine incorporation into RASMCs during the S-phase of the cell cycle. Western blotting analysis revealed that the protein levels of cyclin A, cyclin E, and cyclin-dependent-kinase (CDK) 2 but not cyclin D1 and CDK4 decreased after progesterone treatment, but those of CDK-inhibitory proteins, p21 and p27, increased. Immunoprecipitation showed that the formations of the CDK2-p21 and CDK2-p27 complex were increased and the assayable CDK2 kinase activity was decreased in the progesterone-treated RASMCs. In contrast, the formations of the CDK4-p21 and CDK4-p27 complex and the assayable CDK4 kinase activity were not changed significantly by progesterone treatment. Pretreatment of RASMCs with a p21 or p27 antisense oligonucleotide reduced the progesterone-induced inhibition of [(3)H]thymidine incorporation into RASMCs. In conclusion, these data suggest that progesterone inhibits RASMCs proliferation by increasing the levels of p21 and p27 protein, which in turn inhibit CDK2 kinase activity, and finally interrupt the cell cycle.     

Liver DNA synthesis promoter activity detected in human plasma from subjects with hepatitis

A liver DNA synthesis promoter activity was detected in human plasma from subjects with hepatitis. The assay procedure consisted of intraperitoneal injection into mice of aliquots of plasma, previously chromatographed on Sephadex G-25. After 24 hr, [3H]thymidine was injected and its incorporation into liver DNA measured. The increase in [3H]thymidine uptake of injected mice was not detected in those administered plasma from normal subjects (basal [3H]thymidine incorporation was that corresponding to saline-injected mouse values). At a maximal effective dose (0.3 mg protein per mouse), plasma from subjects with hepatitis increased the mitotic index of mouse liver hepatocytes; at the same dose, plasma from normal subjects had no effect. This DNA synthesis promoter activity appears to be a protein, as it is sensitive to trypsin digestion and heat.     

Ethanol-inhibited [3H]thymidine incorporation via protein kinase C-p44/42 mitogen-activated protein kinase/phospholipase A2 signal pathway in renal proximal tubule cells

BACKGROUND: Ethanol exposure leads to changes of cell proliferation in a variety of cell types. However, how ethanol affects the proliferation of renal proximal tubule cells is not known. METHODS: To examine the effect of ethanol on cell proliferation and its related signaling pathway, [H]thymidine incorporation, release of [H]arachidonic acid (AA), and Western blotting of protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) were performed in primary cultured rabbit renal proximal tubule cells. RESULTS: Ethanol inhibited [H]thymidine incorporation in a time- and dose-dependent manner. An inhibitory effect of ethanol on [H]thymidine incorporation was predominantly observed after 12 hr of treatment with 100 mM ethanol. Ethanol increased AA release and prostaglandin E2 production. In addition, ethanol-induced inhibition of [H]thymidine incorporation was blocked by phospholipase A2 inhibitors and was significantly blocked by PKC inhibitors. Indeed, ethanol induced a PKC translocation from the cytosolic to the membrane fraction. In addition, ethanol-induced inhibition of [H]thymidine incorporation was blocked by PD 98059 (a p44/42 MAPK inhibitor), but not by SB 203580 (a p38 MAPK inhibitor), and ethanol increased the phosphorylation of p44/42 MAPK. Results of phosphorylated p44/42 MAPK by ethanol were consistent with those of [H]thymidine incorporation and [H]AA-release experiments. CONCLUSIONS: Ethanol inhibited [H]thymidine incorporation via PKC, p44/42 MAPK, and phospholipase A2 signaling pathways in primary cultured renal proximal tubule cells.     

Tri-iodothyronine induces proliferation in cultured bovine thyroid cells: evidence for the involvement of epidermal growth factor-associated tyrosine kinase activity

The effects of the tri-iodothyronine (T(3)) secreted by thyroid cells on the growth of the thyrocyte are poorly known. In this study we analyzed the effects of T(3) on the proliferation of bovine thyroid follicles in primary culture previously depleted of endogenous T(3). Cellular deoxiribonucleic acid (DNA) synthesis, determined by [(3)H]thymidine incorporation, was stimulated by T(3) (0.1-5.0 nM) for 24 h in a concentration-dependent fashion with a maximal effect at 1.0 nM T(3) (P<0.01). This T(3) action was time-dependent when assayed from 12 to 72 h. The induction of mitogenic activity was corroborated by the increase in proliferating cell nuclear antigen (PCNA) measured by Western blot analysis. PCNA increased after treatment with T(3) (0.1-5.0 nM) in a concentration-dependent manner. Since T(3) modifies the activity of growth factors whose actions are mainly mediated by tyrosine kinase (TK) activation in diverse cellular types, we assayed the effects of genistein, a general TK inhibitor, and tyrphostin A25, a specific epidermal growth factor (EGF)-receptor (EGFR)-dependent TK activity inhibitor, on the proliferative effects of T(3). The T(3)-induced [(3)H]thymidine incorporation was inhibited by both agents in a concentration-dependent manner. A significant increase in the total TK activity measured in cellular protein extracts was induced by 0.5 and 1.0 nM T(3) (P<0.001). Tyrosine phosphorylation of the EGFR was also stimulated by T(3) (P<0.001) with no change in the EGFR expression as determined by Western blot analysis. Both, the T(3)-stimulated [(3)H]thymidine incorporation and the TK activity were inhibited by a anti-mouse EGF antibody. These results lead us to propose that T(3) could operate as a proliferative agent in bovine thyroid cells through a mechanism involving an autocrine/paracrine EGF/EGFR-dependent regulation.     

Alterations in adipogenic and mitogenic activity of porcine serum in response to hypophysectomy

The importance of the pituitary in postnatal regulation of peripheral preadipocyte proliferation and differentiation was examined by hormone supplementation of hypophysectomized pig serum in primary cultures of preadipocyte and stromal-vascular cells derived from rat inguinal adipose tissue. Hypophysectomized pig serum promoted at least 25% less preadipocyte proliferation, less differentiation of sn-glycerol-3-phosphate dehydrogenase activity, and less histochemical differentiation than serum from intact pigs. Porcine GH supplementation of hypophysectomized serum-stimulated [3H]thymidine incorporation by preadipocytes and stromal cells and also histochemical differentiation of preadipocytes, but not enzymatic differentiation. Insulin-like growth factor I (IGF-I) stimulated [3H]thymidine incorporation by preadipocytes and stromal cells. Enzyme differentiation by developing cells was stimulated by IGF-I. Hydrocortisone supplementation of hypophysectomized serum inhibited [3H]thymidine incorporation and stimulated enzymatic differentiation. Thyroid hormones (T3 and T4) stimulated [3H]thymidine incorporation by preadipocytes in a dose-responsive manner when supplemented to hypophysectomized serum. Thyroid hormones stimulated differentiation of enzyme activity at the lowest concentrations examined. The mitogenic effects of GH, IGF-I, and T4 were not specific to the preadipocyte population, since the stromal-vascular cells responded in a similar manner. However, hypophysectomy resulted in a specific reduction in preadipocyte proliferation while stimulating multiplication of stromal-vascular cells. These results suggest that these hormones are nonspecific mitogens in adipose tissue, while unidentified factors of pituitary origin may be important for the specific regulation of proliferation of preadipocytes. Additionally, hypophysectomy appears to remove mitogenic inhibitors that are specific for the stromal-vascular cells.     

Oestradiol is a potent mitogen and modulator of GnRH signalling in alphaT3-1 cells: are these effects causally related?

GnRH acts via phospholipase C (PLC) activating G-protein coupled receptors to stimulate secretion of gonadotrophins from gonadotrophs. These cells are also regulated by gonadal steroids, which act centrally to influence GnRH secretion, and peripherally to modulate GnRH action. We have shown that oestradiol can stimulate proliferation and modulate GnRH-stimulated [(3)H]inositol phosphate ([(3)H]IP(x)) accumulation (used as a measure of PLC activity) in a gonadotroph-derived cell line (alphaT3-1). Here we show that when alphaT3-1 cells were incubated in medium with 2% foetal calf serum (FCS), [(3)H]thymidine incorporation was not stimulated by oestradiol but was reduced to <2% of control by the oestrogen antagonist, raloxifene. The inhibitory effect of 10 or 1000 nM raloxifene was reversed competitively by oestradiol. A similar pattern of effects was seen when effects of oestradiol and raloxifene on the proportion of cells in the S-phase of the cell cycle (as measured by flow cytometry of propidium iodide-labelled cells) and on oestrogen receptor activity (as measured by trans-activation of the oestrogen-response elements in the vitellogenin promoter) were quantified. In addition, RT-PCR revealed expression of alpha and beta (but not beta2) subtypes of oestrogen receptors. Thus, oestrogen is an essential mitogen for alphaT3-1 cells, its mitogenic effect is oestrogen receptor mediated and is associated with a marked alteration of cell cycle distribution, and the full extent of these effects are best revealed in the presence of raloxifene. Using this strategy, we found that cells cultured for 4 days with 10 nM raloxifene expressed GnRH receptors (K(d) for (125)I-buserelin 4.33 nM) and that their activation by GnRH caused a concentration-dependent increase in [(3)H]IP(x) (in cells labelled with [(3)H]inositol) and inositol 1,4,5 trisphophate (in unlabelled cells). Addition of 10 nM oestradiol (to overcome receptor blockade by raloxifene) reduced GnRH receptor number by 31% but increased maximal effects on [(3)H]IP(x) and Ins(1,4,5)P(3) approximately 4-fold. The effects of oestradiol on GnRH receptor number and signalling were not, however, mimicked by culture for 2 days in medium with 10% FCS and the S-phase blocker, thymidine (15 mM). This treatment increased the proportion of cells in the S-phase 2- to 3-fold but did not alter GnRH receptor number or signalling. Other treatments which altered cell cycle transition (hydroxyurea, colcemid, methotrexate) also failed to alter GnRH receptor number or signalling and no correlation was seen between GnRH receptor number or GnRH-stimulated [(3)H]IP(x) accumulation and the proportion of cells in the S-phase or G2/M-phases of the cell cycle. Thus, oestradiol has pronounced effects on GnRH signalling, proliferation and cell cycle distribution in alphaT3-1 cells, but these trophic effects do not underlie the modulation of GnRH signalling.     

Alteration of Nucleoside Transport of Chinese Hamster Cells by Dibutyryl Adenosine 3′:5′-Cyclic Monophosphate

Cultured Chinese hamster ovary cells showed no significant change in generation time or fraction in the S-phase in the presence of 1 mM N(6),O(2')-dibutyryl adenosine 3':5'-cyclic monophosphate. Growth continued for at least two generations after expression of the morphological transformation induced by this cyclic AMP analog. Despite identical growth rates, apparent rates of DNA and RNA synthesis (incorporation of [(3)H]-thymidine or [(3)H]uridine) were reduced up to 15-fold in log phase by 1 mM cyclic nucleotide. [(3)H]Deoxycytidine incorporation was much less sensitive to dibutyryl cyclic AMP. Uptake studies with [(3)H]thymidine demonstrated an inhibition of transport rate dependent on the concentration of dibutyryl cyclic AMP in the growth medium. The rate of thymidine uptake at 1 degrees was decreased 21-fold by 1 mM cyclic nucleotide; half-maximal inhibition occurred at 6 muM. At 37 degrees , the pool size of acid-soluble thymidylate was strongly reduced by 1 mM cyclic nucleotide, and synergistic reduction of the pool size was found with 0.5 mM aminophylline. Phosphorylation of the acid-soluble intracellular label was unaffected by dibutyryl cyclic AMP. Inhibition of thymidine uptake is attributed to an observed decrease in thymidine kinase activity caused by growth in 1 mM dibutyryl cyclic AMP, and possibly to a simultaneous alteration in membrane permeability. Kinase-facilitated uptake of other metabolites may be regulated in a similar fashion by cyclic AMP.     

Role of cyclic AMP in proliferation of lung tissue in organ culture

The role of cyclic AMP in cell proliferation and division has been the subject of study by a number of investigators in the past 30 years, but the argument of whether cyclic AMP is a negative or a positive regulator has not been settled. We studied the effect of cyclic AMP on proliferation of normal and postpneumonectomized lung tissues in young adult rats by measuring the incorporation of tritiated thymidine into lung DNA in organ culture. In normal lung tissues the incorporation of [3H]thymidine was increased by exogenous dibutyryl cyclic AMP, or by isoproterenol or forskolin to stimulate adenylate cyclase, or by caffeine, which inhibits cAMP phosphodiesterase. The effect of isoproterenol, but not forskolin, was abolished by the beta-adrenergic blocking agent propranolol. The effect of caffeine on [3H]thymidine incorporation was further enhanced in normal lung tissues in the presence of dibutyryl cyclic AMP and in postpneumonectomized lung tissues. Imidazole, a cAMP phosphodiesterase stimulator, also increased [3H]thymidine incorporation in culture, but the effect was not magnified in the presence of exogenous dibutyryl cyclic AMP, nor in postpneumonectomized lung tissues. The data suggest that cyclic AMP acts as a positive regulator in proliferation of lung tissues.     

Nerve growth factor and retinoic acid interactions in the control of small cell lung cancer proliferation

OBJECTIVE: Nerve growth factor (NGF) has antiproliferative and differentiating effects in neuroendocrine tumors. In cell lines derived from small cell lung cancer (SCLC), NGF treatment stimulates NGF receptor expression, activates NGF secretion, inhibits proliferation and abrogates invasion. Since these effects are lost upon NGF withdrawal, it is relevant to identify other differentiation factors that may co-operate with the NGF system to control SCLC growth and differentiation. DESIGN: Retinoic acid (RA), which has been shown to inhibit cell transformation and proliferation, modulates the expression of NGF receptors and the sensitivity to NGF in different cell models. In the present study, we have investigated whether NGF and RA may interact to control the proliferation of SCLC cell lines. METHODS: SCLC cells were exposed to 50 ng/ml NGF or 1 microM all-trans RA for different times. Cell proliferation was measured by the [(3)H]thymidine incorporation test and NGF receptor expression was evaluated by immunofluorescence. RESULTS: We found that RA increased the expression of both trkA and p75 NGF receptors in NCI-N-592 and GLC8 cell lines and prevented the loss of both NGF production and NGF receptor expression occurring when NGF treatment was discontinued. As a result, RA, which did not inhibit the proliferation of untreated cells, abolished NGF withdrawal-related increase in cell proliferation both in vitro and in vivo, thus making permanent the antiproliferative effects of NGF. CONCLUSIONS: These data suggest that combined treatments with NGF and RA or mimicking drugs may represent a strategy to be further investigated for the treatment of SCLC.     

Somatostatin receptor subtypes 2 and 5 differentially affect proliferation in vitro of the human medullary thyroid carcinoma cell line tt

Somatostatin and its receptors (SSTR1 to SSTR5) are expressed in normal human parafollicular C cells and medullary thyroid carcinoma (MTC), but the role of SSTR subtypes in cell growth regulation is still not clear. The present study demonstrates that the human MTC cell line TT stably expresses all the SSTR subtypes and responds to SSTR2 and SSTR5 activation by subtype-selective agonists with two different patterns in terms of [(3)H]thymidine ([(3)H]thy) incorporation and cell number. The SSTR2 preferential agonists (BIM-23120, BIM-23197, BIM-23190, and BIM-23014; 10(-9)-10(-6) M), significantly suppressed [(3)H]thy incorporation (58-13%) and reduced cell proliferation (50-28%), whereas the SSTR5-selective agonist, BIM-23206 (10(-9)-10(-6) M), significantly increased [(3)H]thy incorporation in TT cells (80-175%), but failed to influence cell proliferation. SSTR2 antagonist (BIM-23627) counteracted the action of SSTR2 preferential agonists on TT cells. Furthermore, increasing concentrations of SSTR5-selective agonists, BIM-23206, dose-dependently prevented the suppression of TT cell [(3)H]thy incorporation and proliferation produced by SSTR2 preferential agonist, BIM-23120, showing an antagonism between these compounds. The following conclusions were reached: 1) the human MTC cell line TT expresses all SSTR subtypes; 2) SSTR2 activation inhibits DNA synthesis and cell proliferation, whereas SSTR5 activation increases DNA synthesis; and 3) SSTR2 preferential agonist (BIM-23120) can antagonise SSTR5-selective agonist (BIM-23206) action and vice versa. These findings suggest a tissue-specific function and a tissue-specific interaction between the two receptors.     

Dipeptidyl Peptidase IV Regulates Proliferation of Preglomerular Vascular Smooth Muscle and Mesangial Cells

The purpose of this study was to investigate the role of dipeptidyl peptidase IV in regulating the effects of 2 of its substrates, neuropeptide Y(1-36) and peptide YY(1-36), on proliferation of and collagen production by preglomerular vascular smooth muscle and glomerular mesangial cells from spontaneously hypertensive and normotensive rats. In cells from hypertensive rats, neuropeptide Y(1-36) and peptide YY(1-36) stimulated [(3)H]-thymidine incorporation (cell proliferation index), cell number, and [(3)H]-proline incorporation (index of collagen synthesis); and sitagliptin (dipeptidyl peptidase IV inhibitor) significantly enhanced most of these effects. Neuropeptide Y(3-36) and peptide YY(3-36) (products of dipeptidyl peptidase IV) had little effect on [(3)H]-thymidine incorporation, and sitagliptin did not enhance the effects of either peptide. BIBP3226 (Y(1) receptor antagonist) blocked the effects of neuropeptide Y(1-36) and peptide YY(1-36) on [(3)H]-thymidine incorporation in the absence and presence of sitagliptin. Neuropeptide Y(1-36) and peptide YY(1-36) stimulated [(3)H]-thymidine and [(3)H]-proline incorporation and cell number in cells from normotensive rats; however, the effects were weak and mostly not affected by sitagliptin. Real-time PCR and Western blotting showed similar dipeptidyl peptidase IV mRNA and protein levels in cells from hypertensive versus normotensive rats, with greater levels in smooth muscle versus mesangial cells. Both cell types converted peptide YY(1-36) to peptide YY(3-36) in a concentration-dependent manner that was attenuated by sitagliptin, and dipeptidyl peptidase IV activity was greater in smooth muscle versus mesangial cells. In conclusion, dipeptidyl peptidase IV inhibitors might entail a risk of renal dysfunction because of abnormal proliferation of cells in the preglomerular microcirculation and glomeruli.     

Effect of elastase on aortic smooth muscle cell proliferation

We studied the effects of elastase on [3H]thymidine incorporation into aortic smooth muscle cells. When elastase was added to cultured aortic smooth muscle cells, [3H]thymidine incorporation was inhibited in a dose-dependent manner in the presence of fetal bovine serum. Elastase also inhibited this incorporation in cells treated with epidermal growth factor. Epidermal growth factor (50 ng/ml) stimulated thymidine incorporation, without elastase, but with the addition of 20 units/ml of elastase the incorporation was inhibited 70%. The incorporation of thymidine into cells treated with 50 ng/ml epidermal growth factor was also inhibited 50% by a low concentration of elastase (5 units/ml). These inhibitory effects on thymidine incorporation were also observed in cells stimulated with platelet-derived growth factor. Platelet-derived growth factor (20 units/ml) markedly stimulated thymidine incorporation into cells, and elastase inhibited its activity in a dose-dependent manner. These results suggest that elastase has the potential to prevent the development of atherosclerosis by inhibiting smooth muscle proliferation.     

An antisense oligodeoxynucleotide to growth hormone messenger ribonucleic acid inhibits lymphocyte proliferation

The role of GH in lymphocyte proliferation was studied by examining the effect of an antisense oligodeoxynucleotide (ODN) complementary to GH mRNA. The results of these studies showed that antisense GH ODN treatment inhibits lymphocyte production of immunoreactive GH (irGH). Lymphocytes treated with the GH antisense ODN produced less irGH than did lymphocytes treated with control sense GH ODN. Antisense GH ODN-mediated inhibition of irGH production resulted in a decrease in lymphocyte proliferation. Cells with the antisense GH ODN had less (87%) incorporation of [3H]thymidine [( 3H]TdR) in both resting and Concanavalin-A-stimulated lymphocytes, whereas the incorporation of [3H]TdR in cells treated with a control ODN was not significantly affected. The effect of the antisense ODN on [3H]TdR incorporation was specific, since it could be reversed by hybridization competition with a complementary GH sense ODN or by the addition of exogenous rat GH. Collectively, the data indicate that lymphocytes synthesize and secrete irGH and that irGH produced by these cells can stimulate proliferation, suggesting that GH may play an autocrine/paracrine role in lymphocyte replication.     

Increased thymidine incorporation into fetal rat cartilage in vitro in the presence of human somatomedin, epidermal growth factor and other growth factors

The incorporation of [3H]thymidine by rat costal cartilage in vitro was studied at different fetal and postnatal ages and the effect of partially purified human somatomedin, mouse epidermal growth factor, platelet secretion products, insulin and growth hormone on thymidine uptake by fetal cartilage was examined. Thymidine uptake in plasma-free medium was many times greater in late fetal life than after birth. The incorporation of [3H]thymidine into costal cartilage from 21-day fetuses was significantly (P less than 0.05) increased above control values in the presence of 10 micrograms somatomedin/1, and when cartilage was incubated in medium containing somatomedin and diluted human plasma there was a synergistic action. Epidermal growth factor at a concentration of 1 ng/l was a potent stimulator of thymidine uptake. Secretion products from human platelets after their aggregation by thrombin stimulated [3H]thymidine uptake at a concentration of 2% (v/v), but were inhibitory at high concentrations. High concentrations of platelet secretion products stimulated the incorporation of [35S]sulphate by cartilage. A pharmacological concentration of 10 mu. insulin/ml stimulated [3H]thymidine uptake, but not concentrations of 1 or 100 mu./ml. Growth hormone had no effect. The results showed that fetal cartilage had a greater endogenous mitogenic activity than postnatal cartilage. While somatomedins may be important in the regulation of fetal body growth, other protein growth factors also stimulate fetal skeletal tissues.