Biomedical subjects

L J Murphy

Publications and source records attributed to L J Murphy.

At least 127 records · Page 7Linked to original sources

PubMed1989

Regulation of epidermal growth factor receptor by progestins and glucocorticoids in human breast cancer cell lines.

Human breast cancer cells secrete a number of autocrine peptides which modulate their proliferation rates. The known effects of steroid hormones on breast cancer cell proliferation may be mediated in part by altering the production of these growth factors and/or their interactions with cellular receptor sites. Receptors for epidermal growth factor (EGF), which also bind the autocrine growth factor, alpha-transforming growth factor, are present on a number of breast cancer cell lines and it has previously been shown that T-47D and MCF-7 cells respond to progestins with an increase in the concentration of EGF receptors (EGF-R). In the present study we examined the effects of both progestins and glucocorticoids on EGF binding in 10 human breast cell lines. Five of these lines were progesterone receptor positive and all lines expressed the glucocorticoid receptor (GR). All cell lines were initially incubated for 24 hr with increasing concentrations of the synthetic progestin, medroxyprogesterone acetate (MPA), and the level of specifically bound EGF was determined. An increase in specific binding of EGF was confirmed in two PR-positive lines but, in addition, increases in EGF binding were observed in 4 PR-negative cell lines. In these last lines the synthetic glucocorticoid, dexamethasone, was a more potent inducer of EGF binding than MPA, a known glucocorticoid agonist, while the high-affinity PR ligand, ORG 2058, was without effect. Furthermore, MPA competed with dexamethasone for binding to GR in these cell lines, supporting the view that the induction of EGF binding by MPA in these cells was mediated via the GR. This conclusion was further supported by studies in which addition of the glucocorticoid and progestin antagonist, RU 486, inhibited the effect of ORG 2058 in two cell lines and completely abrogated the effect of dexamethasone in two other lines. Detailed binding studies revealed that the increase in EGF binding was accompanied by an increase in the concentration of EGF-R. This effect was observed when EGF binding was assayed at either 0 degree or 37 degrees C. Further studies demonstrated that the increases in EGF binding following ORG 2058 and dexamethasone treatment were accompanied by increases in EGF-R mRNA levels. Our data illustrate that the binding of EGF by some human breast cancer cells can be regulated by both progestins and glucocorticoids acting via their respective receptors and inducing increases in EGF-R mRNA levels.

Breast Neoplasms↗

PubMed1989

The effect of acute and chronic insulin administration on insulin-like growth factor-I expression in the pituitary-intact and hypophysectomised rat.

Although growth hormone is known to be the main regulator of insulin-like growth factor-I, insulin has also been shown to play a role in regulating serum insulin-like growth factor I levels in diabetic animals. While this effect is thought to be due to correction of metabolic perturbations, some studies have suggest that insulin may have a direct effect on growth and/or insulin-like growth factor-I levels. We have examined the effects of acute and chronic insulin administration to non-diabetic, pituitary-intact and hypophysectomised rats. Rats were injected intraperitoneally with insulin as an acute bolus (10 U) or a chronic subcutanious infusion (low dose; 2.4 U/day, high dose; 12 U/day) over 5 days. Insulin-like growth factor-I mRNA was quantitated by Northern and slot blots of RNA from various tissues. A small (less than 2-fold) but significant increase (p less than 0.05) was seen in hepatic insulin-like growth factor-I mRNA abundance in pituitary-intact rats following acute insulin injection and chronic low dose insulin infusion. An increase in insulin-like growth factor-I mRNA levels was also seen in other tissues including diaphragm, lung, kidney and heart. A significant increase (p less than 0.05) in serum insulin-like growth factor-I levels was also observed 6 h after insulin injection. In contrast, in pituitary-intact rats which received high dose insulin infusion and were hypoglycaemic at the time of death, tissue levels of insulin-like growth factor-I mRNA were reduced compared to saline-treated control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Effects of cycloheximide on hepatic and uterine insulin-like growth factor-I mRNA.

To investigate differences in growth hormone (GH) and estrogen activation of insulin-like growth factor-I (IGF-I) expression, we have examined the effects of cycloheximide on hepatic and uterine IGF-I mRNA abundance in response to GH and 17 beta-estradiol (E2) respectively. In hypophysectomized (hypox) rats a single injection of GH significantly increased hepatic IGF-I mRNA 3.65 +/- 0.68-fold, P less than 0.005, 6 h after injection. Administration of cycloheximide 30 min prior to GH injection completely abolished this response. In contrast, in pituitary intact rats killed 6 h after administration of cycloheximide, hepatic IGF-I mRNA abundance was not significantly different from untreated control rats although the serum IGF-I concentration was significantly reduced; 119.9 +/- 11.8 vs. 270.2 +/- 48.7 ng/ml, P less than 0.005. In immature rats, injection of E2 (1 micrograms/100 g body weight) significantly increased uterine IGF-I mRNA 4.1 +/- 0.4-fold. Cycloheximide did not block the E2-induced increase in IGF-I mRNA but rather significantly enhanced the IGF-I response. These data indicate that continuing protein synthesis is required for GH induction of hepatic IGF-I mRNA in the hypox rat but is not required for E2 induction of uterine IGF-I mRNA. Furthermore, in pituitary-intact rats protein synthesis is not required for maintenance of hepatic IGF-I mRNA levels.

Animals↗

PubMed1989

Scrapie-induced diabetes mellitus in hamsters.

Scrapie-infected hamsters had slightly elevated non-fasting plasma glucose levels, markedly abnormal glucose tolerance tests, and impaired release of insulin in response to a glucose load. Plasma cortisol levels were essentially the same in infected and uninfected animals. Histological examination of the pancreas revealed no morphological changes in infected animals with no alteration in distribution of cells secreting insulin, glucagon and somatostatin. In contrast, brains of scrapie-infected animals had the diffuse vacuolation typical of spongiform encephalopathy. These experiments suggest that scrapie-induced diabetes mellitus in hamsters may result from damage to the central nervous system.

Animals↗

PubMed1989

Dexamethasone inhibits growth hormone induction of insulin-like growth factor-I (IGF-I) messenger ribonucleic acid (mRNA) in hypophysectomized rats and reduces IGF-I mRNA abundance in the intact rat.

Exogenous glucocorticoids are potent inhibitors of skeletal growth in experimental animals and children. The mechanism whereby glucocorticoids exert this effect is unclear, and circulating somatomedin levels have been reported to be low, normal, or high in this situation. Since recent studies have emphasized the importance of autocrine or paracrine insulin-like growth factor I (IGF-I), we have examined the effects of dexamethasone (DXM) on IGF-I gene expression in the hypophysectomized (hypox) and pituitary intact rat. An increase in IGF-I messenger RNA (mRNA) abundance of approximately 5-, 3-, 2-, and 1.5-fold in the liver, proximal tibia, lung, and kidney, respectively, was seen in hypox rats killed 6 h after injection of human GH (100 micrograms/100 g body wt). As little as 1 micrograms/100 g body wt of DXM administered 3 h before GH injection significantly reduced GH induction of IGF-I mRNA in the liver and the proximal tibia (P less than 0.05 and P less than 0.005, respectively). Higher doses of DXM were required to reduce IGF-I mRNA abundance in the kidney and lung. Of the tissues examined the order of sensitivity to DXM was liver greater than tibia greater than kidney greater than lung. In contrast to the effects of DXM on tissue IGF-I mRNA abundance, an approximately 10-fold higher dose of DXM was required to inhibit the GH-induced rise in serum IGF-I concentration. The effect of DXM on steady state IGF-I mRNA abundance in pituitary-intact rats which were killed 9 h after DXM was also examined. The reduction in IGF-I mRNA abundance required higher doses of DXM (6-360 micrograms/100 g body wt) and was less marked in pituitary-intact rats than in GH-treated hypox rats. In the pituitary-intact rats the order of sensitivity to DXM was tibia greater than liver greater than lung greater than kidney. In acute studies serum IGF-I levels were not decreased by any dose of DXM; rather a significant increase in serum IGF-I was apparent in pituitary intact rats, treated with the lowest and highest doses of DXM. When DXM was administered daily for 6 days to pituitary intact rats, body wt gain and hepatic and tibial IGF-I mRNA abundance were significantly reduced. No significant changes were seen in serum IGF-I concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Uterine insulin-like growth factor-I receptors: regulation by estrogen and variation throughout the estrous cycle.

This study was undertaken to identify uterine insulin-like growth factor-I (IGF-I) receptors and examine the regulation of these receptors throughout the estrous cycle and after 17 beta-estradiol (E2) administration to immature rats. We have demonstrated type I IGF receptors in crude uterine membranes by binding and cross-linking experiments. The characteristics of the uterine IGF-I receptor are similar to those reported for other tissues, with a high affinity component (Kd = 0.12 nM; binding capacity = 0.028 pmol/mg protein) and a low affinity component (Kd = 0.98 nM; binding capacity = 0.041 pmol/mg protein). Autoradiographic visualization of [125I]IGF-I binding to uterine sections localized the IGF-I receptors to the smooth muscle cells of the myometrial layer of the uterus, with a higher density of IGF-I receptors in the outer longitudinal layer than in the inner circular layer. In immature rats administration of E2 significantly increased total [125I]IGF-I binding per uterus as early as 6 h after E2 injection. Although [125I]IGF-I binding was significantly increased per mg DNA, because of a more marked increase in membrane protein after E2, [125I]IGF-I binding, when expressed per mg membrane protein, decreased. This change in [125I]IGF-I binding resulted from a change in receptor number with no change in receptor affinity. In mature cycling rats, the proestrous uteri showed the lowest level of [125I]IGF-I binding per mg membrane protein, although because of the greater yield of protein from proestrous uteri, the total [125I]IGF-I-binding capacity of these uteri were greater than that of uteri from other stages of the estrous cycle. The lowest [125I]IGF-I binding was seen in the diestrous uteri. These studies demonstrate the presence of authentic type I IGF-I receptors in the rat uterus localized predominantly to the myometrial smooth muscle cells. In addition, E2 appears to regulate the uterine IGF-I receptor in the immature rat, and the cyclical changes in the mature rat are consistent with a role of E2 in regulation of this receptor in vivo.

Animals↗

PubMed1989

Increased renal aldose reductase activity, immunoreactivity, and mRNA in streptozocin-induced diabetic rats.

Increased accumulation of renal sorbitol has been documented in the diabetic rat, and it has been suggested that this accumulation may be important in the pathogenesis of diabetic nephropathy. It is not clear whether sorbitol accumulation results from increases in substrate, activity of the aldose reductase (AR) protein molecule, or activity due to an increase in the amount of enzyme present. In this study, we have quantitated renal AR activity, immunoreactivity, and mRNA in rats 3 mo after induction of diabetes with streptozocin (STZ-D, 65 mg/kg body wt). Renal AR activity was significantly increased in diabetic rats compared with age-matched nondiabetic controls (0.95 +/- 0.05 vs. 0.51 +/- 0.03 U.mg-1.h-1, respectively, P less than .0005). Western blot analysis demonstrated that the antiserums recognized a single 40,000-Mr protein species in renal homogenates from both diabetic and nondiabetic rats. When quantitated in an immunodot assay, AR immunoreactivity was significantly increased in diabetic rats compared with nondiabetic controls (0.57 +/- 0.03 vs. 0.33 +/- 0.02 U, respectively, P less than .0005). Hybridization of Northern blots with a synthetic 36-nucleotide oligomer and an AR cDNA identified a 1.4-kilobase pair transcript; the abundance of the transcript was significantly increased in poly(A)+ RNA from the kidneys of diabetic compared with nondiabetic rats (P less than .005). This study demonstrates that renal AR activity is increased in the STZ-D rats and suggests that the increased AR activity can be in part explained by enhanced AR gene expression.

Aldehyde Reductase↗

PubMed1988

Epidermal growth factor gene expression in human breast cancer cells: regulation of expression by progestins.

Epidermal growth factor (EGF) is thought to be important in normal mammary development. The presence of EGF receptors in breast cancer cells suggests that it may also have a role in regulating growth of tumors of the human breast. Using a complementary DNA probe for the human EGF precursor we have examined expression of this gene in a series of human breast cancer cells in long term culture. The T-47D cell line demonstrated the highest level of EGF mRNA. EGF expression was not detectable in the MCF-7, BT 20, or HBL 100 cell lines. Surprisingly, in both T-47D and ZR 75 cells, pretreatment with progestins which exert antiproliferative effects under the conditions used increased EGF mRNA levels approximately 6-fold above untreated controls. This effect, demonstrable with as little as 0.1 nM of medroxyprogesterone acetate, was apparent as early as 12 h after addition of progestin and was reversed with the antiprogestin RU 486. Dexamethasone, estradiol, and dihydrotestosterone had no effect on EGF expression in T-47D cells. There was no evidence that the increased levels of EGF mRNA were due to gene amplification. Immunoprecipitation of biosynthetically labeled T-47D conditioned medium with antibodies to human EGF and EGF-precursor revealed the presence of both Mr 40,000 and 18,000 products. Fully processed Mr 6,000 EGF was not detectable in either conditioned medium or cell lysate. These data provide unequivocal evidence for the expression of the EGF gene in some human breast cell lines.

Breast Neoplasms↗

PubMed1988

Cloning and characterization of a cDNA encoding a highly conserved, putative calcium binding protein, identified by an anti-prolactin receptor antiserum.

Using antisera raised against partially purified rabbit mammary gland prolactin receptor, we have isolated a cDNA from a T-47D human breast cancer cell expression library which encodes the putative calcium-binding protein, calcyclin. Since the protein encoded by this cDNA co-purified with the prolactin receptor, we have tentatively named it a prolactin receptor-associated protein (PRA). Hybrid selection and in vitro translation showed that the protein encoded by this cDNA was approximately 10 kDa, a result confirmed by the amino acid sequence derived after DNA sequencing analysis. The PRA gene product has significant sequence similarity to the S-100 proteins, the cystic fibrosis antigen, and p11 proteins. The PRA/calcyclin cDNA obtained from the T-47D human breast cancer cell library has 37 nucleotides more 5'-untranslated information than that of the calcyclin cDNA from human fibroblasts. Since the transcription start site for the calcyclin gene has been confirmed in fibroblasts, our data suggest that a different transcription start site may be used in the T-47D cells. Isolation and nucleotide sequencing of the rat PRA cDNA showed there was 96% predicted amino acid sequence similarity with the human PRA and confirmed the highly conserved nature of the PRA/calcyclin gene between species. The PRA is expressed in most but not all human breast cancer cell lines, e.g. MCF 7. Northern analyses of RNAs from rat tissue indicated that PRA mRNA levels vary widely. They are low in the liver, brain, testes, and ovary, moderate in skeletal muscle, and high in the lung, kidney, and uterus. The availability of prolactin receptor-positive human breast cancer cell lines, which do (T-47D) and do not (MCF 7) express the PRA/calcyclin gene, will allow the investigation of the role, if any, of PRA in prolactin action.

Amino Acid Sequence↗

PubMed1988

Progestin regulation of EGF-receptor mRNA accumulation in T-47D human breast cancer cells.

Incubation of T-47D human breast cancer cells with the synthetic progestin, medroxyprogesterone acetate (MPA), resulted in a time and dose-dependent increase in epidermal growth factor-receptor (EGF-R) mRNA. Concentrations of MPA as low as 1 nM resulted in a greater than five fold increase in EGF-R mRNA. A significant increase (2-3 fold) in EGF-R mRNA was apparent 12 hr after exposure to MPA and a further increase was seen 12-48 hr after addition of MPA to the cultures. From these studies it is concluded that the increased EGF binding in progestin-treated T-47D cells results at least in part from increased EGF-R gene expression. We believe this is the first report of a steroid hormone modulating expression of this growth factor receptor gene.

Antineoplastic Agents↗

PubMed1988

Impaired estrogen-induced uterine insulin-like growth factor-I gene expression in the streptozotocin diabetic rat.

Circulating somatomedin-C/insulin-like growth factor-I levels are low in the diabetic rat and unresponsive to exogenous growth hormone. However, the nature of this defect in growth hormone action remains unclear and there is little data on insulin-like growth factor-I gene expression in response to other stimuli and in non-hepatic tissues where insulin-like growth factor-I may have important paracrine and/or autocrine actions. We have previously shown that 17-beta estradiol stimulates uterine insulin-like growth factor-I expression in the ovariectomised rat. In this report uterine and hepatic insulin-like growth factor-I gene expression have been examined in the streptozotocin-diabetic rat. Serum insulin-like growth factor-I concentrations were significantly reduced in diabetic rats compared to normal rats (0.72 +/- 0.08 vs 1.23 +/- 0.05 U/ml, p less than 0.0005) and hepatic insulin-like growth factor-I mRNA abundance was similarly reduced in diabetic rats to 49 +/- 5% of that seen in non-diabetic intact rats (p less than 0.005). In contrast, uterine insulin-like growth factor-I mRNA abundance was not significantly reduced in diabetic rats compared to control rats (76 +/- 12%, p = NS). Although both diabetic and non-diabetic rats demonstrated a significant increase in uterine wet weight following a single injection of 17-beta estradiol the increase in uterine insulin-like growth factor-I expression was significantly less marked in diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Differential effects of estrogen and growth hormone on uterine and hepatic insulin-like growth factor I gene expression in the ovariectomized hypophysectomized rat.

The acute and chronic effects of 17 beta-estradiol (E2) and GH on uterine and hepatic insulin-like growth factor I (IGF-I) gene expression in ovariectomized hypophysectomized (ovx-hypox) rats were examined. Six hours after a single injection of E2 (5 micrograms/100 g BW), uterine IGF-I gene expression was increased 22.5 +/- 5.4-fold (P less than 0.005) above that in untreated rats. In the same experiment E2 alone had no significant effect on hepatic IGF-I gene expression. Similarly, in chronic experiments uterine IGF-I in ovx-hypox rats receiving 0.1 or 1 microgram/rat.day E2 for 10 days was significantly increased compared to that in ovx-hypox rats that did not receive E2 [5.38 +/- 0.79 vs. 1.10 +/- 0.15 (P less than 0.005) and 6.64 +/- 0.28 vs. 0.93 +/- 0.06 (P less than 0.005), respectively]. While administration of human GH alone significantly increased uterine IGF-I expression (3.76 +/- 1.61-fold compared to that in untreated rats; P less than 0.05), a significant and reproducible attenuation of E2-induced IGF-I expression was seen in the two acute experiments where GH reduced the E2-induced response by 36 +/- 3.7% and 53 +/- 19.4%. While chronic administration of E2 to ovx-hypox rats resulted in uterine growth, a significant decrease in body weight was seen in rats treated with 1 microgram/day E2 compared to that in untreated ovx-hypox controls (-4.3 +/- 1.5 vs. 2.5 +/- 0.6 g; P less than 0.0005). E2 treatment also significantly decreased the GH-induced increase in weight gain at each GH dose by approximately 40%. GH-induced hepatic IGF-I gene expression and serum IGF-I concentration were similarly reduced by chronic E2 administration. In contrast, in acute experiments where E2 alone had no effect on hepatic IGF-I expression, it acted in a synergistic fashion with GH and resulted in significantly greater accumulation of IGF-I mRNA. From these observations we conclude that 1) both E2 and human GH are potent stimulators of IGF-I gene expression in appropriate target tissues; and 2) in addition to any effects E2 has on GH secretion and IGF-I action, the growth-retarding effect of estrogen in the rat involves inhibition of GH-dependent hepatic IGF-I expression.

Animals↗

PubMed1988

Stimulation of hepatic insulin-like growth factor-I gene expression by ovine prolactin: evidence for intrinsic somatogenic activity in the rat.

The effect of ovine PRL (oPRL) on hepatic insulin-like growth factor I (IGF-I) gene expression was examined in the hypophysectomized (hypox) rat and compared with that seen in hypox rats treated with ovine GH (oGH) and human GH (hGH). A single injection of oPRL (100 micrograms/100 g BW) resulted in a 15-fold increase in hepatic IGF-I messenger RNA abundance 12 h after administration. Serum IGF-I increased from 82.9 +/- (SE) 9.0 to 137.0 +/- 15.4 ng/ml. In acute studies, where the slopes of 4-point dose-response curves were compared, oPRL was approximately 50% as potent as oGH in stimulating serum IGF-I and hepatic IGF-I messenger RNA accumulation. In chronic experiments, where a single daily injection of oPRL (0, 10, 30, or 90 micrograms/day) was administered for 10 days, a significant increase in body wt was observed at each dose. However, in contrast to hypox rats which received oGH, no dose-response relationship was apparent, and oPRL was less than 10% as potent as oGH in this regard. A small but significant increase in serum IGF-I and hepatic IGF-I expression was observed in hypox rats chronically injected with oPRL when killed 6-7 h after the final injection. However, by extrapolation from the hGH dose-response curves, oPRL was 6.8% and 22.7% as potent as hGH in stimulating serum IGF-I and hepatic IGF-I accumulation respectively. The observations reported here demonstrate that oPRL has significant somatogenic activity in the rat as determined by the three variables measured. Whereas oPRL had significant effects on body weight gain, serum IGF-I and hepatic IGF-I expression, the potency of oPRL by comparison with the two other somatogenic hormones was more marked in acute experiments compared to the effects seen after chronic administration. These results suggest that oPRL, like human GH, has both somatogenic and lactogenic activity in the rat and therefore may be an inappropriate control in studies of the effects of somatogenic hormones in the rat.

Animals↗

PubMed1987

Methionine oxidation in human growth hormone and human chorionic somatomammotropin. Effects on receptor binding and biological activities.

The oxidation of the methionine residues of human growth hormone (hGH) and human chorionic somatomammotropin (hCS) to methionine sulfoxide by hydrogen peroxide has been studied. The kinetics of oxidation of individual methionine residues has been measured by reverse-phase high pressure liquid chromatography tryptic peptide mapping. Met-170 is completely resistant to oxidation in both hormones. The other 3 methionine residues in hCS (Met-64, Met-96, and Met-179) have markedly different reaction rates. Oxidation of the methionine residues does not appear to cause gross conformational changes in either hGH or hCS, as judged by CD and 1H NMR spectroscopy. Oxidation of Met-14 and Met-125 in hGH has little effect on affinity of the hormone for lactogenic receptors or on its potency in the Nb2 rat lymphoma in vitro bioassay for lactogenic hormones. The oxidation of Met-64 and/or Met-179 in hCS reduces profoundly both its affinity for lactogenic receptors and its in vitro biological potency. It is inferred by induction that residues 64 and/or 179 are critical for the binding of both hGH and hCS to lactogenic receptors and the expression of lactogenic biological activity.

Animals↗

PubMed1987

Tissue distribution of insulin-like growth factor I and II messenger ribonucleic acid in the adult rat.

The insulin-like growth factors (IGFs) have both metabolic and growth-promoting activities in many cell and tissue types. Although the IGFs are present in serum, they are also thought to have important autocrine and paracrine functions. Using complementary DNA (cDNA) probes for rat IGF-I and mouse IGF-II, we have investigated the tissue distribution of messenger RNAs (mRNAs) for these growth factors in adult rats. IGF-I cDNA hybridized with three groups of transcripts, 7.0, 1.8 and 0.7-1.1 kilobases, which were detectable in all tissues examined, with liver demonstrating the highest level of expression. IGF-II cDNA also hybridized to a number of mRNAs, the most abundant of which was 4.0 kilobases. Of the tissues examined, IGF-II expression was highest in the brain, barely detectable in the liver, and undetectable under the conditions used, in lung, ovary, testes, and mammary gland. These studies support the notion of paracrine or autocrine function for IGF-I and demonstrate tissue-specific IGF-II expression in the adult rat.

Animals↗

PubMed1987

Growth hormone stimulates sequential induction of c-myc and insulin-like growth factor I expression in vivo.

The effect of GH administration to hypophysectomized rats on expression of the c-myc proto-oncogene and insulin-like growth factor I (IGF-I) in the liver and kidney was examined. In both tissues maximal expression of c-myc occurred by 1 h after a single injection of human GH (100 micrograms/100 g bw). In the liver the maximal c-myc mRNA level was increased 12 +/- 3.9-fold (mean +/- SEM; n = 4), while the maximal c-myc level in the kidney was increased 3.4-fold (n = 2) compared to levels in basal hypophysectomized rats. In both the liver and kidney the IGF-I cDNA hybridized under stringent conditions to three transcripts with apparent sizes of 7.0, 1.8, and diffuse group of transcripts of 0.7-1.1 kilobases. Each of these transcripts demonstrated some degree of GH dependence. Under the hybridization condition used, the 7.0-kilobase IGF-I mRNA was virtually undetectable in the hypophysectomized control rat liver, while the smaller transcripts were easily detectable. Peak expression of each of the IGF-I transcripts occurred 6-12 h after GH administration. Maximal IGF-I expression in the kidney occurred 9 h after the GH injection. To determine whether the increase in c-myc expression following GH could result from a small but undetectable increase in IGF-I expression in these tissues, we administered human recombinant IGF-I to hypophysectomized rats (50 micrograms/100 g bw, ip). Despite a significant increase in serum IGF-I concentrations to levels greater than those present in the first 3 h after GH administration, no increase in c-myc expression was apparent in either the liver or kidney. These observations suggest that GH itself, rather than IGF-I, initiates the mitogenic response in the liver and kidney that follows GH administration to hypophysectomized rats.

Animals↗

PubMed1987

Estrogen induction of N-myc and c-myc proto-oncogene expression in the rat uterus.

The mechanisms involved in the proliferative response of the uterus to estrogen are poorly understood. The c-myc proto-oncogene has recently been shown to be rapidly activated in quiescent cells exposed to various mitogens. We have examined expression of c-myc and a closely related proto-oncogene, N-myc, in the rat uterus after in vivo administration of 17beta-estradiol (E2), 5 micrograms/100 g body weight, to prepubertal ovariectomized rats. Maximal c-myc messenger RNA (mRNA) accumulation, as determined by densitometric analysis of Northern blots of poly (A)+ uterine RNA was observed 3 h after E2 treatment. Maximal expression of c-myc was 8.6 +/- 0.8-fold (mean +/- SEM for 3 separate experiments) compared to basal levels seen in vehicle-treated ovariectomized rats. The maximal level of c-myc mRNA in the E2-stimulated uterus was higher (3- to 6-fold) than that observed in uteri from intact rats in either diestrous or the proestrous-estrous stages of the estrous cycle. There was no significant difference in the level of uterine c-myc mRNA throughout the estrous cycle. Under stringent conditions, the N-myc DNA probe hybridized with a single 3 kilobase (kb) transcript which was virtually undetectable in ovariectomized rat uteri and increased 6-fold within 15 min after E2 treatment. Maximal induction was seen 30-60 min post E2 treatment. At 1 h post E2 the level of N-myc mRNA was 9.3 +/- 0.4-fold (n = 3) compared to vehicle-treated rats. Under conditions of slightly reduced stringency, N-myc DNA also hybridized with a 2.2 kilobase transcript. Expression of the N-myc related gene also occurred more rapidly after E2 administration than c-myc mRNA. Our in vivo data are analogous to the in vitro observations that mitogen stimulation of quiescent cells results in a rapid accumulation of myc proto-oncogene mRNAs. In cycling cells in vitro and in the uterus of intact rats throughout the estrous cycle, the level of expression of the myc oncogenes is relatively constant. Since expression of the c-myc and N-myc proto-oncogenes appears to be restricted to different cell and tissue types our data indicate that there is at least one cell type present in the quiescent uterus that is able to respond rapidly to E2. The rapidity of the N-myc response would argue for a direct effect of E2. In contrast the c-myc response is considerably delayed and may be mediated via autocrine, paracrine, or circulating estrogen-dependent growth factors.

Actins↗

PubMed1987

Identification, characterization, and regulation of a rat complementary deoxyribonucleic acid which encodes insulin-like growth factor-I.

A complementary DNA (cDNA) encoding rat insulin-like growth factor 1 (IGF-I) has been isolated from a kidney cDNA library. By analogy with the sequence of human and mouse preproIGF-IA this cDNA encodes rat preproIGF-I from amino acid minus 3 to amino acid 105. The predicted protein sequence shows 96% and 99% homology with the human and mouse preproIGF-I, respectively. Under stringent conditions the rat IGF-I cDNA hybridizes with at least three mRNA, messenger RNA species which have apparent sizes of 7, 1.8, and 0.7-1.1 kilobases. All three IGF-I transcripts are detectable in each of the normal rat tissues examined and the relative order of abundance in tissues from intact adult male rats is liver greater than lung greater than kidney greater than thymus greater than spleen greater than heart greater than skeletal muscle (quadriceps femoris) greater than testes greater than brain. The GH dependence of the IGF-I mRNAs was demonstrated by the administration of a single ip injection of human GH (hGH) to hypophysectomized (hypox) rats which resulted in an increase in all three transcripts in each of the tissues examined. The increase in IGF-1 mRNAs was most marked in skeletal and cardiac muscle (9.7- and 9.5-fold compared to hypox controls, respectively) and least marked in the brain. In the liver only a 4-fold increase in IGF-I expression was observed, possibly because of the relatively high level of IGF-I expression in the tissue in the hypox control rats. Each of the IGF-I messenger RNAs appeared to increase in parallel and the time course of IGF-I induction was similar in each tissue with maximal levels of IGF-I transcripts present 6 to 12 h after GH administration. A dose-dependent increase in IGF-I mRNAs was observed in most tissues of hypox rats treated for 10 days with hGH. Significant correlations between growth, as determined by body weight gain and IGF-I expression were observed in liver (r = 0.97), kidney (r = 0.90), quadriceps femoris (r = 0.95), diaphragm (r = 0.92), and thymus (r = 1.0). The IGF-I mRNA levels in tissues from rats that had been treated with the highest dose of hGH (90 microgram/rat X day) were similar to those observed in normal intact rats. This study confirms the highly conserved nature of the IGF-I precursor and provides clear evidence for the GH dependence of IGF-I gene expression in multiple tissues of the rat.

Amino Acid Sequence↗