Tumor necrosis factor stimulates DNA synthesis in the liver of intact rats

TNF is cytotoxic to tumor cell lines but enhances growth of some nontransformed cells. Because animals administered TNF have an increase in liver size, we studied the [3H]thymidine incorporation into DNA in the liver of intact rats. A significant increase in [3H]thymidine incorporation is seen 20 hours following TNF administration and peaks at 24 hours. The lowest dose of TNF that increases DNA synthesis is 10 micrograms/200 g rat with a maximal increase occurring with 25 micrograms/200 g, considerably less than the dose required for maximally increasing plasma triglycerides. The increase in [3H]thymidine incorporation was shown to be due to an increase in DNA polymerase alpha activity (associated with the replication of DNA) rather than DNA polymerases beta (associated with DNA repair) plus gamma activity. These results indicate that TNF administration stimulates DNA replication in the liver of intact animals.     

Pituitary hormones regulate c-myc and DNA synthesis in lymphoid tissue

Hypophysectomy of Fischer 344 rats of both sexes led to a rapid involution of the thymus and spleen which was associated with a profound decrease in spontaneous DNA synthesis in these organs. The proportion of B lymphocytes in the spleen, of T cells and their subsets (CD4+/CD8+) in spleen and thymus, and the histological structure of the involuted organs remained normal. Treatment of hypophysectomized animals with growth hormone (GH) or prolactin (PRL) stimulated the expression of the c-myc proto-oncogene and DNA synthesis and reversed the involution in these organs. Replacement doses of adrenocorticotrophic hormone, follicle-stimulating hormone, luteinizing hormone, or thyroid-stimulating hormone had no influence on thymus or spleen size and DNA synthesis. A rapid expression of c-myc was also observed in thymuses and spleens of intact rats after the injection of GH or PRL. In vitro physiological concentrations (2.5 ng/ml) of either ovine or rat PRL or GH stimulated the incorporation of [3H]thymidine by thymus and spleen cells. These results indicate that GH and PRL regulate lymphocyte growth. This regulatory role is likely to serve as the principal mechanism of immunoregulation by these hormones.     

Cytofluorometry of lymphocytes infected with Epstein-Barr virus: effect of phosphonoacetic acid on nucleic acid.

DNA synthesis in Epstein-Barr virus (EBV)-infected lymphocytes was inhibited by phosphonoacetic acid (PAA) as measured by [3H]thymidine incorporation. PAA, at a concentration of 200 microgram/ml, inhibited [3H]thymidine incorporation by human umbilical cord lymphocytes infected with EBV strain P94 but had little effect on DNA synthesis in mitogen-stimulated cells. Transformed cell lines did not develop from infected cord cell cultures treated with 100 microgram of PAA per ml. Cytofluorometric analysis showed marked increases in cellular nucleic acid content (RNA plus DNA) as early as 9 days after infection of cord cells in the absence of PAA and before significant enhancement of [3H]thymidine incorporation became apparent. Moreover, EBV led to increases in cellular nucleic acid even when 200 microgram of PAA per ml was added to cell cultures before infection. The apparent discrepancy between results obtained by [3H]thymidine incorporation and cytofluorometry is explained either by significant inhibition of cellular DNA polymerases by PAA or by a block at the G2 + M phase of the cell cycle. The data suggest that EBV initiates alterations in cellular nucleic acid synthesis or cell division without prior replication of viral DNA by virus-induced DNA polymerases.     

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.     

Stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the gastrointestinal tract of the suckling mouse

The effects of epidermal growth factor (EGF), cortisone and thyroxine on deoxyribonucleic acid (DNA) synthesis in the esophagus, stomach, small intestine and colon have been studied in suckling mouse. Daily administration of EGF [4 micrograms/g body weight (bw)/day] during 3 days to 8-day-old mice induced a significant increase of the incorporation of [3H]thymidine into DNA in the stomach, the small intestine, and the two halves of the colon. The DNA synthesis in the esophagus remained unaffected by the EGF treatment. The maximal increase of [3H]thymidine incorporation into DNA was observed in the colon, and represented 112%. Daily administration of cortisone acetate (25 micrograms/g bw/day) or thyroxine (1 microgram/g bw/day) during 3 days to 8-day-old mice had no significant influence of the DNA synthesis of any part of the gastrointestinal tract. These results show that EGF is able to affect the DNA synthesis in the stomach, small intestine and colon of suckling mice.     

Regulation of growth hormone mRNA synthesis by 3,5,3'-triiodo-L-thyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells). Dissociation between nuclear iodothyronine receptor concentration and growth hormone mRNA synthesis during the deoxyribonucleic acid synthesis phase of the cell cycle

3,5,3'-Triiodo-L-thyronine (T3) regulates the growth rate and GH production of cultured GC cells, a rat pituitary tumor cell line. We have previously demonstrated a parallel increase in cellular content of DNA and nuclear T3 and glucocorticoid receptors during the DNA synthesis (S) phase of the GC cell growth cycle. To determine the relationship between the increase in nuclear hormone receptors and GH production in S-phase cultures, we measured the synthesis rate of GH by pulse-labeling with [3H]leucine and immunoprecipitation as well as the relative concentration of GH mRNA by dot hybridization employing formaldehyde-treated cytoplasm and GH cDNA. Total protein synthesis was similar in S-phase and asynchronous cultures. However, in comparison to asynchronous cultures, S-phase cells had an increased GH synthesis rate, p less than 0.005 (from 13,430 +/- 609 to 19,150 +/- 1160 cpm/10(6) cells/2 h) and increased GH mRNA, p less than 0.001 (from 7.2 +/- 1.2 to 14.5 +/- 1.5 relative A units). The S-phase-associated augmentation in GH production did not appear to result from a decrease in ADP-ribosylation induced by 2 mM thymidine treatment which was utilized for the S-phase synchronization. To determine whether increased GH mRNA and GH synthesis in S-phase was associated with an increase in synthesis of GH mRNA, we measured the incorporation of [3H]uridine into GH mRNA by incubating partially synchronized S-phase cells with [3H]uridine and isolating 3H-labeled GH mRNA by hybridization to GH cDNA immobilized on nitrocellulose filters. Total RNA synthesis was similar in asynchronous, S-phase and G1 cell populations. However, the mean incorporation of [3H]uridine into GH mRNA of S-phase cultures was decreased to 52, 59, and 61% (counts/min of GH mRNA/10(6) cells), 49, 59, and 65% (ppm of total RNA), and 64 and 69% (ppm of poly(A)+ RNA) of asynchronous cultures. Our studies show further that the decrease in [3H]uridine incorporation into GH mRNA did not result from a cell cycle specific change in efficiency of hybridization or exclusively to an S-phase associated increased rate of degradation of GH mRNA. Thus, despite increased nuclear T3 and glucocorticoid receptors and, increased GH mRNA and GH synthesis, the synthesis rate of GH mRNA appears decreased in S-phase GC cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of growth hormone on the in vitro maturation of fetal islets

To study the effects of growth hormone (GH) on the in vitro maturation of fetal islets, the fetal islets were cultured for 7 days in RPMI 1640 containing 10% fetal bovine serum and 11.1 mM glucose with or without GH. Culture with 1 microgram/ml of bovine GH increased the DNA content of the islets and [3H]thymidine incorporation into DNA confirming results of other investigators. In addition, however, the insulin secretory dynamics and ultrastructural morphometrics were investigated. It was found that GH-treated islets demonstrated increased insulin release during acute glucose stimulation when expressed as microunits per islet per minute. However, when insulin release during acute glucose stimulation was expressed as microunits per microgram of DNA per minute to compensate for the increased DNA content of GH-treated islets, no change in insulin release was observed compared to control islets. When GH-treated islets were perifused with a linear glucose gradient, the insulin secretory response was suppressed as indicated by changes in the threshold level, plateau level, and half-maximal response. Ultrastructural morphometric data showed that the average beta-cell volume in control and GH-treated islets was the same, eliminating the possibility that beta-cell hypertrophy occurred. Similarly, the nuclear volumes of the beta cells in control and GH-treated islets remained unchanged. This finding coupled with the observed increased DNA content and [3H]thymidine incorporation suggests that GH functions by increasing cell multiplication within the islets and not by inducing polyploidy. Finally, the volumes of cytoplasmic organelles in control and GH-treated islets were the same indicating that cytodifferentiation did not occur.     

Bovine placental lactogen stimulates DNA synthesis of bovine mammary tissue maintained in athymic nude mice

Mammary tissue from five midpregnant heifers was transplanted subcutaneously into ovariectomized athymic mice (eight pieces/mouse). After a recovery period of 19 days, mice were injected daily for 5 days with buffer (50 mM NH4HCO3, pH 7.8) as control, 17 beta-estradiol (1 micrograms) plus progesterone (1 mg). Concurrently with the buffer or steroid hormone injections, mice were injected with bovine placental lactogen (0, 5, or 25 micrograms), bovine prolactin (0, 3.4, or 17.2 micrograms), or bovine growth hormone (0, 3.4, or 17.2 micrograms). All mice were injected with 2-bromo-alpha-ergocryptine (0.1 mg/day). Transplanted bovine mammary tissue was incubated for 4 hr in minimum essential medium containing 1 mu Ci/ml [3H]TdR. Two pieces were processed for autoradiography and the others were used for DNA assay and total [3H]TdR uptake. Bovine placental lactogen, prolactin, and growth hormone each increased [3H]TdR incorporation into DNA in a linear, dose-response manner. Addition of ovarian steroids to bPL resulted in a significant increase over protein hormones alone. Autoradiographic analysis indicated that the observed differences in DNA synthesis were due to hormonal effects on epithelial, rather than stromal, DNA synthesis. These results provide the first evidence of a mammogenic role of bovine placental lactogen.     

Nerve growth factor-induced increase in [3H]thymidine incorporation into pancreas of young rats and its partial blockade by propranolol or partial sialoadenectomy

Administration of nerve growth factor (NGF) twice daily for 2 days to rats 11 days at the time of the initial injection resulted in a 6.6-fold increase in [3H]thymidine levels of pancreas, when comparison was made to levels of untreated controls. Isoproterenol (ISO), a beta-adrenergic agonist known to produce marked increase in [3H]thymidine incorporation into DNA of salivary glands, caused increases in levels of [3H]thymidine in pancreas that were similar in magnitude to those induced by NGF. The combined administration of ISO and NGF did not cause any increase above those observed with either agent alone. Administration of propranolol (3 mg/kg body wt) prior to administration of ISO prevented the usual ISO-induced increase in DNA synthesis, but propranolol in either a 3- or 9-mg/kg body wt dose, caused only a 50% inhibition of NGF-induced thymidine incorporation. In the absence of the submandibular-sublingual glands, the ISO failed to induce the usual high levels of thymidine incorporation, whereas NGF induced the same high levels observed in rats with submandibular glands intact. NGF did not alter the distribution of beta 1- and beta 2-adrenoceptors in the pancreas but did increase norepinephrine when the initial administration was at age 5 days, but not when it was given at age 10 days. Since NGF increased DNA synthesis in the absence of submandibular-sublingual glands, whereas ISO did not, this suggests that ISO requires NGF to induce beta 1-activation and subsequent synthesis and that NGF is a direct activator.     

Growth hormone stimulates the peripheral conversion of thyroxine into triiodothyronine by increasing the liver 5'-monodeiodinase activity in the fasted and normal fed chicken

Normal fed and 2 days fasted Warren chickens were injected intravenously with 100 micrograms of ovine growth hormone (GH) and ovine prolactin and plasma concentrations of thyroid hormones were assayed prior and up to 2 h after injection. Fasting alone decreases T3, but increases T4. An injection of GH resulted in increases of plasma T3 concentrations in two fasting experiments by 40% (after 3/4 h) and 104% (after 1 h). In normal fed animals no increase is observed in the first experiment, whereas a 35% increase occurs in the second one. An injection of 100 micrograms prolactin does not influence T3 in normal fed or fasting animals. Both GH and prolactin, however, may decrease plasma concentrations of T4. In a separate experiment 50 micrograms and 200 micrograms of GH raised the decreased T3 levels after fasting by 39% and 60% respectively 1 h after injection and by 24 and 61% respectively in normal fed chicken, whereas prolactin was ineffective in this regard. Using Hisex chickens, the influence of an injection of 100 micrograms GH on plasma concentrations of thyroid hormones could be confirmed. At the same time GH increases the liver 5'-monodeiodinase activity by 330% after 1 h and by 147% after 2 h. The peroxidase activity is not influenced in normal fed chickens, but GH decreases this activity in food deprived animals after 1 h and 2 h. It is concluded that ovine GH, but not prolactin, stimulates the peripheral conversion of T4 into T3 in both normal fed and food deprived chicken and that this effect is dose dependent.     

Sertoli cell-secreted protein(s) stimulates DNA synthesis in purified rat Leydig cells in vitro

We have examined the effects of Sertoli cell-secreted proteins (SCSP) on [3H]thymidine incorporation by purified preparations (greater than 96%) of rat Leydig cells to determine whether Sertoli cells influence DNA synthesis in these cells in vitro. Incubation of Leydig cells isolated from testes of rats of ages 16 to 90 days with SCSP (Mr greater than 10,000) induced significant dose-, time- and age-related increases in [3H]thymidine incorporation by the cells. A dose-response curve to SCSP showed that as little as 0.2 micrograms SCSP/ml consistently induced a small but significant increase (31% and 10% above control; P less than 0.001) in [3H]thymidine incorporation by Leydig cells isolated from immature (26 days) and mature (70 days) rats, respectively. The maximum response (230% and 48% above control) was obtained with a concentration of 18 micrograms SCSP/ml in cells isolated from immature and mature rats, respectively. Hydroxyurea, a specific inhibitor of replicative DNA synthesis, significantly (P less than 0.001) inhibited both basal and SCSP-induced [3H]thymidine incorporation in Leydig cells from immature and adult rats without affecting the viability of the cells. Incubation of immature rat Leydig cells in SCSP for 48 h also stimulated a 3-fold increase in cell number. The component of the crude SCSP which stimulated Leydig cell [3H]thymidine incorporation is trypsin-sensitive, heat-stable, and adsorbs to a heparin-agarose affinity column but not to concanavalin A-Sepharose. The secretion of this factor(s) by Sertoli cells is stimulated independently by FSH and testosterone. These results demonstrate for the first time that cultured Sertoli cells secrete a protein(s) which, in vitro, stimulates rat Leydig cell replicative DNA synthesis.     

Effect of estradiol on the early incorporation of (3H) thymidine into uterine DNA on immature mouse.

The incorporation of [3H]thymidine into uterine DNA was markedly depressed within 10 to 30 minutes after intraperitoneal administration of 17beta-estradiol to immature mouse. Maximum inhibition occurred about 6 hours after the hormone was administered. Uterine DNA content and the amount of [3H]thymidine incorporated into the acid-soluble fraction was not affected during the period of hormone-induced inhibition. Moreover, the in vitro incorporation of [3H]thymidine by isolated estradiol-treated mouse uterus was blocked. In contrast to the uterus, 17beta-estradiol did not influence the incorporation of thymidine into mouse liver DNA. Evidence is presented to show that the incorporation of thymidine into uterine DNA was blocked initially by 17beta-estradiol.     

Variations in prolactin and growth hormone production during cellular growth in clonal strains of rat pituitary cells.

A permanent, clonal strain of rat pituitary tumor cells (GH3-cells) spontaneously synthesizes and secretes prolactin (rPRL) and growth hormone (rGH) into the culture medium. The rates of hormone production (microng extracellular hormone/mg cell protein/24 hours) and synthesis (vida infra) as well as the rate of [3H]thymidine incorporation into DNA (DNA synthesis) have been studied. During logarithmic growth rPRL and rGH production increased to 160 and 250% of the value at day 2 after plating, while during the plateau phase of cell growth hormone production decreased to initial values. The fluctuations in rPRL production could be fully explained by variations in the rate of rPRL synthesis: [3H]eucine incorporated into rPRL as measured with immunoprecipitation and polyacryl-amide gel electrophoresis. Also the rates of synthesis and production of rGH showed parallel changes during exponential and plateau phase of growth, but this hormone was probably degraded intracellularly. The relative reduction in the rate of synthesis of rPRL and rGH during the plateau of growth corresponded closely to the fall in the rate of DNA synthesis. The reduction in rPRL synthesis could not be explained through an inhibition by extra-cellular rPRL accumulation or by cell to cell interaction occurring in dense cultures. The intracellular concentrations of both hormones were unaltered during logarithmic growth, but rose to 500% for rPRL and 200% for rGH during the plateau phase. In spite of the marked variations in basal rPRL and rGH production the GH3 cultures of different ages were equally able to increase rPRL and decrease rGH production in response to thyrotropin releasing hormone (3 X 10(-7) M) and 17beta-estradiol (10(-8)M).     

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

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.     

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.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

THYMIDINE METABOLISM AND DEOXYRIBONUCLEIC ACID SYNTHESIS IN THE DEVELOPING RAT BRAIN

—In growing rat brain, the specific activity of DNA at 12 h after the subcutaneous injection of [³H]thymidine underwent a sharp rise during the first 6 days of life, dropping just as precipitously by 15 days, thereafter continuing to decrease with increasing age. When [³H]thymidine was given to 6-day-old rats, a considerable amount was taken up immediately into the brain. Thymidine taken up into the acid-soluble fraction was readily phosphorylated to its nucleotides, thymidine mono-, di-, and triphosphate (TMP, TDP and TTP) within only 30 min following injection. The highest specific activity was found in TTP. The incorporation of of [3H]thymidine into DNA took place over a longer period of time after injection.     

Evidence for a direct trophic effect of bombesin on the mouse pancreas: in vivo and cell culture studies

The present work studied the effect of chronic bombesin on the mouse pancreas and analyzed whether or not this effect was direct. Bombesin administered s.c. 3 times daily for 4 days at various concentrations (0.1, 1, 10, 20 micrograms/kg b. wt.) induced pancreatic growth in a dose-dependent manner. This growth was characterized by an increase in pancreatic weight, its protein and RNA contents suggesting cellular hypertrophy. Pancreatic enzyme content was also increased, especially for amylase (14-fold) and at a lesser degree for chymotrypsin and lipase (2.5-fold). The DNA content of the gland increased significantly after a 1 microgram/kg bombesin treatment suggesting hyperplasia. [3H]thymidine incorporation into DNA increased slightly from 24 h after the first bombesin injection and more obviously at 72 and 96 h indicating DNA synthesis. To determine the direct effect of bombesin on pancreatic acinar cell growth cells were cultured as monolayers on collagen gels in media lacking added hormones and containing 2.5% FBS with or without bombesin (1 microM-1 nM) or caerulein (10 nM). [3H]thymidine incorporation into DNA was increased by caerulein (10 nM) and bombesin (100 nM and 1 microM). Therefore, it is concluded that bombesin is a pancreaticotrophic peptide in mice. Moreover, it is suggested that this effect occurs directly on pancreatic cells.     

Direct evidence that the insulin receptor mediates a mitogenic response in cultured human fibroblasts

To define the role of the insulin receptor in mediating a mitogenic response in cultured human fibroblasts, the effects of specific monoclonal antibodies against the insulin and the type I IGF receptor on insulin-stimulated [3H]thymidine incorporation were investigated. Insulin stimulated [3H]thymidine incorporation in a biphasic fashion. In the first phase, a half-maximal effect was observed at 20 ng/ml, and a seemingly maximal effect was obtained at 100-1000 ng/ml. With 10 micrograms/ml insulin, a secondary increase in [3H]thymidine incorporation was seen which was similar to the maximal effect of IGF-I. These [3H]thymidine incorporation results were corroborated with cell replication studies. MC-51, a highly specific monoclonal antibody for the insulin receptor, inhibited the stimulation of [3H]thymidine incorporation by 25 ng/ml of insulin. AlphaIR-3, a monoclonal antibody specifically directed against the type I IGF receptor, had no significant effect on insulin-stimulated [3H]thymidine incorporation at low (10-1000 ng/ml) concentrations of insulin. However, alpha IR-3 interfered with the incremental increase in [3H]thymidine incorporation observed at 10-100 micrograms/ml insulin. These data demonstrate that insulin, at low concentrations, is capable of stimulating DNA synthesis and replication of human fibroblasts through interaction with its own receptor, while at supraphysiological concentrations, much of insulin's mitogenic effect is mediated through the type I IGF receptor.