Estradiol stimulates c-myc proto-oncogene expression in normal human breast epithelial cells in culture.

The proto-oncogene c-myc is involved in the stimulation of cell proliferation, and its expression is known to be stimulated by estradiol (E2) in human breast cancer cell lines and various non-cancerous E2-dependent tissues. However, little information is currently available concerning its expression and regulation in normal human breast tissue. We therefore studied c-myc expression and hormone modulation in normal human breast epithelial (HBE) cells in culture, routinely obtained in our laboratory and which remain hormone-dependent. On these normal HBE cells, E2 induced a biphasic increase in c-myc mRNA level, with a first peak as early as 30 min, and a secondary increase after 2 h of treatment; this stimulation was dose-dependent, with an optimal concentration of 10 nM E2. Its primary action is probably at the transcriptional level since the half-life of c-myc mRNA measured in the presence of actinomycin D (12 +/- 3 min) was not modified by E2 treatment. In addition, E2 stimulation of c-myc mRNA does not require protein synthesis since it was not suppressed by cycloheximide treatment. Western blot studies of c-myc protein in HBE cells revealed the same biphasic pattern of stimulation, with a first peak after 60 min and a second one after 2 h of E2 treatment. In conclusion, the c-myc proto-oncogene is expressed in normal HBE cells, as in breast cancer cells. Moreover, E2 stimulates c-myc expression which, therefore, may partly mediate the growth-promoting effect of E2.

Estradiol-induced down-regulation of estrogen receptor. Effect of various modulators of protein synthesis and expression.

Incubation of MCF-7 cells with estradiol (E2) down-regulates estrogen receptor (ER) resulting in a progressive reduction of the capacity of cells to concentrate selectively [3H]E2. Scatchard plot analysis failed to detect any transformation of residual receptors into peptides of lower binding affinity. [3H]Estrone gave an identical ER disappearance pattern with an ER half-life comprised between 2 and 3 h. A similar value was established by incubating the cells with [3H]tamoxifenaziridine ([3H]TAZ) for 1 h before the addition of excessive unlabeled E2 which induced ER-down regulation and impeded any further labeling of the residual receptors. Submission of the [3H]TAZ labeled cell extracts to SDS-PAGE revealed no progressive emergence of low molecular weight cleavage products of the receptor (< 67 kDa). Two inhibitors of protein kinases, H-7 at 40 microM and H-89 at 20 microM, failed to block the E2-induced ER down-regulation. On the contrary, the protein phosphatases 1 and 2A inhibitor, okadaic acid, was effective with concentrations higher than 0.1 microM indicating that a dephosphorylation mechanism was involved in this phenomenon. Cycloheximide (CHX) also significantly reduced the receptor decrease at concentrations higher than 1 microM. G-C specific intercalating agents [actinomycin D (AMD) and chromomycin A3 at 1 microM] also prevented ER disappearance; ethidium bromide (EB) and quinacrine were ineffective. AMD and CHX operated immediately after their addition to the medium indicating an inhibitory action on the synthesis of an RNA and/or a peptide with high turnover rate involved in ER decline. Moreover, AMD produced its suppressive effects under conditions impeding any labeling of newly synthetized receptors (i.e. [3H]TAZ with an excess of unlabeled E2) rejecting the possibility of an increasing ER production which may partially hamper its disappearance. Finally, E2-induced ER mRNA down-regulation was similarly abolished by AMD while EB and CHX were devoid of effect.

Expression of the c-erbB2 gene in the BT474 human mammary tumor cell line: measurement of c-erbB2 mRNA half-life.

BT474 and SK-BR-3 mammary adenocarcinoma cells contain eight copies of the c-erbB2 gene but overexpress the mRNA 80 times over the levels measured in normal breast or in the HBL-100 cell line. Using Northern blot analysis and molecular titration based on RNAase protection assay, the decrease in the c-erbB2 mRNA level was monitored in BT474 cells treated with actinomycin D from 1 up to 24 h. The c-erbB2 degradation rate during the first 12 h corresponds to a calculated c-erbB2 mRNA half-life of approximately 7 h. Forty percent of the mRNA present in the cells before treatment remains undegraded after transcription has been blocked for 24 h. Pretreatment with cycloheximide results in complete mRNA degradation in 24 h, suggesting that labile proteins stabilize part of the c-erbB2 mRNA population. Comparison with the c-erbB2 mRNA turnover in HBL-100 'normal' cells indicated that the accumulation of the c-erbB2 gene product in the tumor cells is not the result of stabilization of the messenger. Rather, it is correlated with an increased rate of c-erbB2 mRNA transcription as indicated by run-on transcription assays. Both BT474 and SK-BR-3 tumor cell lines were found to synthesize 20-40 times more c-erbB2 mRNA than HBL-100 cells.

Insulin-like growth factor II in two human colon-carcinoma cell lines: gene structure and expression, and protein secretion.

Abnormal expression and structural modification of the IGF-II gene in correlation with high IGF-II production have recently been described in human colorectal tumors in comparison with normal adjacent tissues. Here, we have studied IGF-II in 2 human colon-carcinoma cell lines, HT29 and COLO 320DM. RFLP analyses revealed no apparent alteration at the IGF-II locus in these 2 cell lines. HT29 cells weakly expressed IGF-II mRNA in comparison with the high over-expression previously observed in some colorectal tumors, and only the 4.8-kb mRNA species was present. In addition, the serum-free medium conditioned by HT29 cells contained high IGF-II levels (approx. 900 ng/10(8) cells), as measured in a specific IGF-II radioimmunoassay (RIA). After chromatography on Bio-Gel P-60, we observed that 64% of the total IGF-II secreted by HT29 cells were present as a high-molecular-weight form of about 17 kDa. In contrast, no detectable expression of the IGF-II gene was observed in COLO 320DM cells, and low IGF-II levels were secreted by these cells in the serum-free medium, with only 9% total IGF-II represented by the large species.

Stimulatory effect of oestradiol-17 beta and tamoxifen on gross cystic disease fluid protein 15,000 production and mRNA levels in T47D human breast cancer cells.

Oestradiol-17 beta and tamoxifen regulate the synthesis of a gross cystic disease fluid protein (GCDFP-15) in T47D human breast cancer cells. Dose-response curves of GCDFP-15 mRNA contents and GCDFP-15 levels in culture media and cells versus hormone or antihormone concentration have been established. Production of GCDFP-15 was increased by oestradiol-17 beta, tamoxifen and 4-OH tamoxifen. The effect of tamoxifen and 4-OH tamoxifen was greater than the effect of oestradiol-17 beta. Moreover, oestradiol-17 beta and 4-OH tamoxifen acted synergystically in enhancing GCDFP-15 release. The strong oestrogenic effect of the antioestrogen tamoxifen in regulating GCDFP-15 may reflect an unusual interaction between the tamoxifen-oestrogen receptor complex and the DNA oestrogen-responsive elements. As oestrogen control of GCDFP-15 depends also on the cell line studied, investigation of GCDFP-15 could extend our knowledge of the possible mechanism of action of oestrogens or antioestrogens.

Tumor IGF-II content in a patient with a colon adenocarcinoma correlates with abnormal expression of the gene.

We have recently reported abnormal insulin-like growth factor-II (IGF-II) mRNA levels in a number of human colorectal adenocarcinomas. Using an IGF-II radioimmunoassay, we have now detected high levels of both 10-kDa and 7.5-kDa IGF-II species (2,370 ng/g) in a right colon tumor showing a 800-fold IGF-II gene over-expression in comparison to the normal adjacent tissue. The higher-molecular-mass form represents 74% of the total immunoreactive IGF-II detected in the tumor. This form appears to be less reactive in the radioreceptor assay than in the radioimmunoassay. The insulin-like growth factor-I (IGF-I) concentration in the tumor is low. The patient's pre-operative serum IGF-II level is not increased and the proportion of the 10-kDa species is normal. In addition, the IGF-II/IGF-I ratio is 3 in the serum and 308 in the tumor. Our results show that the very high IGF-II level produced by the tumor does not influence the seric concentration of the growth factor.

Abnormal expression and structural modification of the insulin-like growth-factor-II gene in human colorectal tumors.

The insulin-like growth factor II (IGF-II) is a small protein implicated in fetal growth and development. It may play a role in the neoplastic process. The IGF-II gene is located on the short arm of chromosome II near insulin and c-Ha-ras I genes. Three distinct promoters control the transcription of this gene, leading to different IGF-II mRNA species. We have analyzed 21 human colorectal tumors and found overexpression of IGF-II in 6 of them (30%). When compared with expression in normal adjacent tissues, IGF-II mRNA increase in these tumors was either moderate (2- to 15-fold) or very marked (200- to 800-fold). In situ hybridization experiments confirmed that high IGF-II mRNA amounts were localized in cancer cells of the tumors overexpressing the IGF-II gene. In addition, DNA analysis revealed a structural modification of one IGF-II locus in one tumor characterized by very high IGF-II mRNA.

Paracrine action of transforming growth factors.

Polypeptide growth factors form a class of regulatory molecules which exert their effects by binding to specific receptors present on the cell surface. Most of the time the exact role of these factors in the healthy body is unknown. Some, like PDGF and TGF beta, seem to be involved in wound healing. Others, like EGF, promote epithelial cell growth and differentiation. The site of synthesis of most polypeptide growth factors is unknown. Their target can be identified by detecting the cells which present the specific receptors at their surface. It is though that polypeptide growth factors have a paracrine mode of action. Many different cancerous cells produce polypeptide growth factors and the appropriate receptors. Thus, they are able to stimulate their own growth in an autocrine fashion. Recently, some polypeptide growth factors and receptor genes or cDNAs have been molecularly cloned. Growth factor genes and messengers are much more complex than would be expected from the size of the polypeptide. Some cDNAs have been introduced into bacterial expression vectors and large amounts of the factors have been produced by bacteria. New tools, such as molecular probes and specific antibodies, are thus now available to investigate the production of the growth factors and their receptors. The same tools will facilitate the identification and understanding of the molecular mechanism whereby cancerous cells produce the growth factors and the appropriate receptors simultaneously. The importance of growth factors and receptors in cancer is stressed by the finding that three oncogenes are in fact the genes coding for one growth factor and two receptors. Finally, the molecular probes and the specific antibodies raised against these molecules can be used to identify precisely the growth factor(s) and receptor(s) produced abnormally in cancers. Antibodies that inhibit specifically the interaction of this very growth factor with its receptor could then be developed, thus allowing human tumour cell growth to be controlled.

Oncogene involvement in radiation- and virus-induced mouse osteosarcomas.

Internal irradiation of mice using bone seeking radionuclides results in the activation of endogenous retroviruses and in the subsequent development of bone tumors. Genomic DNA from an osteosarcoma cell line, derived from an 90Sr-induced bone tumor, was cotransfected with the plasmid pSV2-neo into NIH/3T3 cells and G418-resistant transfectants gave rise to colonies in soft agar. Southern blot analysis of these first cycle transformants revealed the presence of extra copies of c-ras. We have analysed the arrangement of ecotropic murine leukemia proviral sequences in seven 90Sr-induced bone tumors and one osteosarcoma cell line of CF1-mice. Integration of ecotropic and/or ecotropic recombinant proviruses seems to be involved in rearrangements of 3' provirus cellular junction fragments occurring in all tumor DNAs analysed, but no indication for site-specific integration was found. We also determined the primary structure of FBR-MuSV, a transforming retrovirus able to induce bone tumors in newborn mice. FBR-MuSV contains sequences from all four exons of the murine c-fos gene, but lacks sequences encoding the first 24 and the last 98 amino acids of the c-fos gene product. The coding region of FBR-MuSV has also undergone two small in frame deletions. Thus, the v-fosFBR-MuSV retains 236 amino acids of the 380 amino acids of the murine c-fos product. In FBR-MuSV-transformed cells two fos-containing mRNAs have been detected: a 3.3-kb full-size genomic RNA and a 2.2-kb subgenomic mRNA as revealed by both fos- and MuLV-hybridization probes.

Mutagenic activity of non-volatile organic matter associated with suspended matter in urban air.

Extracts of atmospheric suspended matter showed a direct mutagenic effect in the Ames test. This effect was increased by metabolic activation. These extracts were separated into an aliphatic, an aromatic and a polar fraction. The aliphatic fraction had no effect, with or without activation; the aromatic fraction showed the greatest mutagenicity with and without activation; the polar fraction was also mutagenic, but its metabolic activation did not enhance the effect. The active compounds in this last fraction could be represented in a significant part by oxygenated derivatives of polycyclic aromatic hydrocarbons.

Ascorbic acid effect on methylcholanthrene-induced transformation in C3H10T1/2 clone 8 cells.

Ascorbic acid (50 micrograms/ml), added to the culture medium on a biweekly basis, suppresses the methylcholanthrene-induced transformation of C3H10T1/2 cells.