Overexpression of the c-erbB-2 protein in human breast tumor cell lines

The c-erbB-2 proto-oncogene is amplified in a high percentage of primary human breast tumors, suggesting that the overexpression of this gene may be involved in the development of human breast cancer. We have investigated five human breast tumor cell lines and have detected amplified c-erbB-2 gene copies in two of them. This amplification leads to overexpression of the c-erbB-2 protein. In addition, two other cell lines have elevated protein levels without gene amplification, suggesting that other mechanisms can lead to overexpression of the c-erbB-2 protein. These results are similar to those that we obtained during a study of primary breast tumors (Berger et al.: Cancer Res 48:1238-1243, 1988). These breast tumor cell lines should be useful for an analysis of c-erbB-2 expression and of the mechanisms that in some cases lead to overexpression.     

The pR UV+ plasmid, transfected into mammalian cells, enhances their UV survival.

It has been recently reported that the pR plasmid enhances the UV survival in E.coli c600. In order to test whether this function may be expressed also in mammalian cells, LTA (tk- aprt-) mouse cells were cotransformed with pR plasmid DNA and ptk1 plasmid as selectable marker. Tk+ transformants were analyzed for their UV survival and for the presence of pR DNA sequences by blot-hybridization. The results show a correlation between the enhanced UV survival and presence of pR DNA sequences in cotransformed LTA mouse cells.     

The effect of intracellular oxygen on the metabolization of Benzo(a)pyrene and Benzo(k)Fluoranthene. A microspectrofluorometric analysis in single living cells

Summary The fluorescence increase which accompanies the injection of glycolytic intermediates to Benzo(a)pyrene (BP) and Benzo(k) Fluoranthene-B(k)F treated EL2 ascites cancer cells, under aerobic and anaerobic conditions, has been studied in a microspectrofluorometer. In the carcinogen-treated cells the altered fluorescence increase pattern (in reference to control cells) which is observed at aerobiosis and attributed to BP or B(k)F metabolization, is not any more observable at anaerobiosis, in which case the fluorescence increase of the carcinogen-treated cells resembles that of the controls. This difference in behavior is discussed and a comparison is initiated between the response to injection in cells treated with BP (compound with K region) or B(k)F (compound without K region).     

Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular clones.

Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.     

Epidermal growth factor receptor, platelet-derived growth factor receptor, and c-erbB-2 receptor activation all promote growth but have distinctive effects upon mouse mammary epithelial cell differentiation.

Three different receptor tyrosine kinases, epidermal growth factor (EGF), c-erbB-2/neu, and platelet-derived growth factor (PDGF) receptors, have been found to be present in the mouse mammary epithelial cell line HC11. We have investigated the consequences of receptor activation on the growth and differentiation of HC11 cells. HC11 cells are normal epithelial cells which maintain differentiation-specific functions. Treatment of the cells with the lactogenic hormones glucocorticoids and prolactin leads to the expression of the milk protein beta-casein. Activation of EGF receptor has a positive effect on cell growth and causes the cells to become competent for the lactogenic hormone response. HC11 cells respond optimally to the lactogenic hormone mixture and synthesize high levels of beta-casein only if they have been kept previously in a medium containing EGF. Transfection of HC11 cells with the activated rat neuT receptor results in the acquisition of competence to respond to the lactogenic hormones even if the cells are grown in the absence of EGF. The activation of PDGF receptor, through PDGF-BB, also stimulates the growth of HC11 cells. Cells kept only in PDGF do not become competent for lactogenic hormone induction. The results show that activation of the structurally related EGF and c-erbB-2/neu receptors, but not the PDGF receptor, allows the HC11 cells to subsequently respond optimally to lactogenic hormones.     

Diminished serotonin uptake in platelets of transgenic mice with increased Cu/Zn-superoxide dismutase activity.

Reduced levels of the neurotransmitter serotonin in blood platelets is a clinical symptom characteristic of individuals with Down's syndrome. To investigate the possible involvement of the Cu/Zn-superoxide dismutase (CuZnSOD) gene, which resides at the Down locus on chromosome no. 21, in the etiology of that symptom, we examined blood platelets of transgenic mice harboring the human CuZnSOD gene. It was found that platelets of transgenic CuZnSOD animals, which overexpress the transgene, contain lower levels of serotonin than nontransgenic littermate mice, due to a reduced rate of uptake of the neurotransmitter by the dense granules of the platelets. We found that the pH gradient (delta pH) across the dense granule membrane, which is the main driving force for serotonin transport, was diminished in dense granules of transgenic-CuZnSOD. Furthermore, a significantly lower than normal serotonin accumulation rate was also detected in dense granules isolated from blood platelets of Down's syndrome individuals. These findings suggest that CuZnSOD gene dosage is affecting the dense granule transport system and is thereby involved in the depressed level of blood serotonin found in patients born with Down's syndrome.     

The structure of the human liver-type phosphofructokinase gene

We have isolated the gene for the human liver-type phosphofructokinase, from upstream to the 5' mRNA terminus to beyond the polyadenylation site. The gene is at least 28 kb long and is divided into 22 exons; it contains conventional splice-junction sequences and one polyadenylation signal. Exons and introns are quite rich in G and C residues; some 60% of all nucleotides are either G or C. Five possible sites of polymorphism have been found. The gene structure reveals no signs of internal similarities despite protein sequence evidence which suggests that the PFK molecule is divided into two similar halves. The structure and organization of the human liver-type PFK gene are shown to be extremely similar to those of the rabbit muscle-type PFK.