Role of growth factor synthesis in the acquisition of insulin/insulin-like growth factor I independence in rat mammary carcinoma cells.

In previous work, we demonstrated that a subset of carcinogen-induced rat mammary carcinomas consists of cells that are independent of growth factors strictly required by normal rat mammary epithelial (RME) cells for long-term growth in serum-free medium. Furthermore, only those tumors that expressed growth factor independence in vitro were serially transplantable in vivo. The present studies were aimed at determining if the independence of insulin (IN)/insulin-like growth factor I (IGF-I) is mediated by autocrine factors synthesized by the rat mammary tumor (RMT) cells. The results of these experiments indicate that IN/IGF-I-independent RMT cells do not synthesize IGF-I that is detectable at the message or peptide level. Both normal and neoplastic cells do, however, secrete IGF-I-binding activity. Conditioned medium, cell lysates, and cell extracts obtained from growth factor-independent cells do not contain growth factor activity that can substitute for IN for growth of IN-dependent RMT cells. Growth factor-independent cells do not express a density dependence for growth in IN-free medium nor do they respond to exogenous IN or IGF-I in low density growth assays. By contrast, growth factor-dependent cells that were rendered IN/IGF-I independent by transfection with an expression vector containing the IGF-I gene secrete IGF-I-like biological activity that is readily detectable and maintain responsiveness to exogenous IN at low densities. Taken together, these results suggest that growth factor-independent RMT cells are truly autonomous of IN/IGF-I for growth and are not synthesizing a growth factor that satisfies their IN/IGF-I requirement in an autocrine manner.

1,25-dihydroxyvitamin D3, transforming growth factor beta1, calcium, and ultraviolet B radiation induce apoptosis in cultured human keratinocytes.

Apoptosis is a cellular process of self-directed suicide that plays a key role during morphogenesis and in the maintenance of homeostasis in continuously renewing tissues. Currently, apoptosis is detected mainly by gel electrophoresis of fragmented DNA and by typical ultrastructural features such as cell shrinkage and chromatin condensation. Recently, an in situ technique was developed that allows the detection of the apoptotic process in cells and the quantitation of apoptosis in cell populations. We applied this technique to evaluate the apoptotic process in cultured normal human keratinocytes under basic conditions and after stimulation with factors and agents that are presumed but have never been proved to induce apoptosis in these cells. Apoptosis was analyzed after stimulation with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], transforming growth factor beta1 (TGFbeta1), calcium, UVB, or tumor necrosis factor alpha (TNFalpha). All these factors except TNFalpha induced apoptosis in human keratinocytes. Whereas UVB and calcium were good apoptogenic stimuli at 6 and 24 h, respectively, the vitamin D derivative and TGFbeta1 induced apoptosis after 5 and 6 d in culture. Apoptosis was also established by DNA fragmentation and electron microscopy. Finally, TUNEL technique showed that the number of apoptotic cells increases slightly (5-10%) from 24 to 144 h even in untreated keratinocytes. Our studies indicate that factors normally involved in the regulation of cell growth and differentiation can also control apoptosis.

Communication: expression of the novel inhibitor of apoptosis survivin in normal and neoplastic skin.

Apoptosis plays a fundamental part in epidermal homeostasis, and apoptotic cells have been detected in normal and diseased skin. Little is known, however, on the inhibitory mechanisms of apoptosis at the skin level. In addition to bcl-2, a novel inhibitor of apoptosis designated survivin and structurally analogous to IAP apoptosis inhibitors has been recently identified. The expression of survivin in normal and pathologic skin was investigated. Immunohistochemical studies revealed that survivin is expressed in basal keratinocytes, but not in suprabasal epidermal layers, with a pattern similar to bcl-2. In western blots, the anti-survivin antibody recognized a single band of 16.5 kDa in protein extracts from normal human keratinocytes in culture, in agreement with the predicted size of survivin. In addition, survivin immunoreactivity was detected in benign and malignant melanocytic lesions, with strong expression in invasive lesions of melanomas. Whereas survivin staining was undetectable in benign epithelial tumors, such as seborrheic keratoses, it was observed in all epidermal layers in Bowen's disease. Interestingly, at variance with bcl-2, survivin was markedly expressed in squamous cell carcinoma, but virtually lacking in basal cell carcinoma, suggesting that these two apoptosis inhibitors may act through different anti-apoptotic pathways. Deregulation of survivin may influence both epidermal homeostasis and the development of melanoma and nonmelanoma skin cancer.

Growth of only highly tumorigenic cell lines is inhibited by EAP, a human placental fraction.

We have previously shown that a fraction from a human placental extract, EAP, inhibited growth in soft agar of a human lung squamous adenocarcinoma cell line, A-2182, and of Ha-ras oncogene-transfected murine BALB/c 3T3 cells. We report here the activities of this extract on several cell lines which have different degrees of transformed phenotype. Human esophagus and colorectal cell lines were derived from tumors at different stages of neoplasic progression, and murine BALB/c 3T3 cells were transfected with various oncogenes. In all three models, growth of the most highly tumorigenic cells was inhibited by the presence of EAP in soft agar medium, while growth of non- and low tumorigenic counterparts was not affected or was stimulated by the placental extract. In addition, EAP did not significantly affect the doubling time of anchorage-dependent cell growth, suggesting that EAP specifically suppresses tumorigenic characteristics of cells such as their ability to grow in soft agar medium. These effects appear to be in contrast to those of transforming growth factor beta, which exerts its most profound effect on less tumorigenic cells.

Comparative effects of insulin and refeeding on DNA synthesis, HMP shunt and cholesterogenesis in diabetic and fasted rats.

DNA synthesis, cholesterogenesis and the enzymes of the hexosemonophosphate (HMP) shunt pathway were investigated in liver of diabetic rats treated with insulin and in fasted/re-fed rats. Both insulin and refeeding were found to induce liver cell proliferation, accompanied by a remarkable increase in cholesterogenesis. An enhancement of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (G6PD) activities was also found in insulin-treated diabetic rats and in re-fed rats, supporting the concept that these two enzymes are involved in the proliferative process. Since insulin did not exert the same biochemical effects in a non replicating cell population, such as in insulin-treated normal rats, these studies provide new evidence of a close correlation between DNA, cholesterol synthesis and HMP shunt enzymes during cell proliferation.

Hexose monophosphate shunt and cholesterogenesis in lead-induced kidney hyperplasia.

De novo cholesterol synthesis and hexose monophosphate (HMP) shunt were studied in rat kidney stimulated to proliferate by a single administration of lead nitrate. Lead-treated rat kidneys showed an increase in DNA synthesis, as measured by [3H]thymidine incorporation starting at 18 h and with a maximum at 24 h. Renal DNA synthesis was preceded by an increase in de novo cholesterol synthesis and an enhancement in the activity of the HMP shunt, as indicated by increased activity of G6PDH and 6PGDH. These findings indicate that enhancement of cholesterol synthesis and of the HMP shunt is closely associated with the active proliferative process induced in the kidney by treatment with lead nitrate.

The role of Ha-ras oncogenes in growth factor independence in rat mammary carcinoma cells.

To determine if activation of the c-Ha-ras-1 gene is involved in the acquisition of growth factor independence in 7,12-dimethylbenz[a]anthracene (DMBA)--and N-nitrosomethylurea (NMU)--induced rat mammary carcinomas, three strategies were used. First, Ha-ras DNA from growth factor-independent DMBA-induced rat mammary tumor cells was amplified using the polymerase chain reaction and examined for the presence of mutations in the first and second exons of Ha-ras-1 by restriction fragment length polymorphism analysis, allele-specific oligonucleotide hybridization, and direct sequencing. No mutations were found in the codon 12/13 or codon 61 regions of the Ha-ras-1 gene. Second, a similar analysis of an NMU-induced mammary carcinoma showed that it harbored an activating mutation in codon 12 of Ha-ras-1. When analyzed for growth factor requirements, these cells were found to express limited growth potential in all media tested, in contrast to growth factor-independent cells, which proliferated extensively in the presence or absence of exogenous growth factors. Third, growth factor-dependent rat mammary tumor cells and spontaneously immortalized rat normal mammary epithelial cells were transfected with an activated form of the Ha-ras-1 (T24) gene, and the growth factor requirements of the transfected cells were examined. The ras-transfected cells retained the growth factor requirements of the normal cells. In addition, ras-transfected cells were transplanted into syngeneic rats and nude mice, and no tumors developed after 6 mo in vivo. These results indicate that, in rat mammary tumor cells, neither growth factor independence in vitro nor transplantability are directly mediated by Ha-ras oncogenes. The results also suggest that ras activation and growth factor independence may be associated with independent pathways to malignancy in rat mammary tumorigenesis.

Structure of the murine Mac-2 gene. Splice variants encode proteins lacking functional signal peptides.

The murine Mac-2 gene is composed of six exons dispersed over 10.5 kilobases. S1 nuclease mapping showed multiple transcription initiation sites, clustered within a 30-base pair region. Sequence analysis revealed that a consensus initiator sequence is located in this area which lacks a TATA motif. The untranslated first exon contains an alternative splice donor site, confirming the existence of two cDNA species with the potential to encode proteins differing at their NH2 termini. In vitro expression and translocation experiments demonstrate that both of the alternatively spliced variants of Mac-2 encode proteins which lack a functional signal peptide. Subcellular fractionation studies indicate that most of the Mac-2 protein is present in the cytosol. These results support the view that Mac-2 is exported from the cell by an unusual mechanism which does not depend on the presence of a signal peptide.

Hepatic glucose-6-phosphate dehydrogenase, cholesterogenesis, and serum lipoproteins in liver regeneration after partial hepatectomy.

Liver regeneration after partial hepatectomy was used as an experimental model for studying mammalian cell division and replication. The rate of cell proliferation in this hyperplastic model was correlated with hepatic de novo synthesis of cholesterol, with the hexose monophosphate shunt pathway of glucose metabolism, and with serum lipoproteins. An increase of hepatic cholesterol esters and of incorporation of tritiated water in cholesterol esters was observed at 24 hr after partial hepatectomy. Partial hepatectomy also resulted in an increase of hepatic glucose-6-phosphate dehydrogenase and in alteration of serum lipoproteins, primarily due to a selective decline in high density lipoprotein fraction.

Hexose monophosphate shunt and cholesterol synthesis in the diabetic and fasting states.

The livers of streptozotocin-induced diabetic and fasted rats showed a decreased cholesterol synthesis measured by in vitro incorporation of [2-14C]acetate. A significant decrease of glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD), and pyruvate kinase (PK) was also observed 7 days after administration of streptozotocin. These enzymatic activities were also low in livers of 72 hr fasted animals. An increase of glucose-6-phosphatase (G-6-Pase) was observed consistently in diabetic as well as in fasted rats. Suitable amounts of insulin and refeeding normalized the alterated enzymatic activities in diabetic and in fasted animals, respectively.

Enhancement of cholesterol synthesis and pentose phosphate pathway activity in proliferating hepatocyte nodules.

The endogenous synthesis of cholesterol in hepatocyte nodules, induced in male Wistar rats, by a single dose of the hepatocarcinogen diethylnitrosamine followed by a selection procedure, was investigated and was compared with that in surrounding and control tissue. In addition, the activity of enzymes related to carbohydrate metabolism (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose-6-phosphatase and pyruvate kinase), was measured. Hepatocyte nodules showed a striking increase in their capacity for synthesizing cholesterol, in comparison to surrounding and control tissues, and an enhancement in the activity of the pentose phosphate pathway, as indicated by increased activity of glucose-6-phosphate dehydrogenase and of 6-phosphogluconate dehydrogenase, and a concomitant decrease of glucose-6-phosphatase. The stimulation of cholesterol synthesis and of the pentose phosphate pathway was associated with increased incorporation of labelled thymidine into DNA. These data indicate that, among other metabolic disturbances, enhancement of cholesterol synthesis and of the pentose phosphate pathway, is accompanied by an increased proliferative capacity of hepatocyte nodules.

HMP-shunt and cholesterol metabolism in experimental models involving normal and preneoplastic liver growth.

Previous studies from our laboratories have shown a stimulation of HMP-shunt, cholesterol metabolism and DNA synthesis during cell proliferation. In order to understand the co-ordinated regulation of these pathways during cell growth, the above metabolic pathways were studied in: liver regeneration after partial hepatectomy, lead-induced liver hyperplasia, liver cell proliferation induced by insulin in streptozotocin-diabetic rats, liver cell proliferation in fasted rats after refeeding and, hepatocyte nodules induced by a selection procedure. The results indeed indicate that changes in HMP-shunt and cholesterol metabolism occur at a very early stage during the process of normal as well as preneoplastic cell growth. The coordinated regulation between cell growth and changes in these metabolic pathways needs further study.

A new set of monoclonal antibodies directed to proline-rich and central regions of p53.

The p53 protein can adopt several conformations in cells--"latent," "active," or mutant--depending on cellular stress or mutations of the TP53 gene. Today, only a few antibodies discriminating these conformations are available. We produced three new anti-p53 monoclonal antibodies (MAbs) directed against epitopes of human p53. The H53C1 MAb recognizes an epitope located at the N-terminal part of the central region of p53 and can discriminate mutant from wild-type conformation. The H53C2 and H53C3 MAbs are against different epitopes within the proline-rich region of p53. Moreover, the H53C2 epitope is located in the second negative regulatory domain of p53 between residues 80 and 93. These MAbs can be used as new tools to study and modulate the cellular functions of p53.