Expression of the rat aldolase B gene: a liver-specific proximal promoter and an intronic activator

The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.     

Proteins encoded by the human c-myc oncogene: differential expression in neoplastic cells.

To examine myc protein products in the wide variety of human tumor cells having alterations of the c-myc locus, we have prepared an antiserum against a synthetic peptide corresponding to the predicted C-terminal sequence of the human c-myc protein. This antiserum (anti-hu-myc 12C) specifically precipitated two proteins of 64 and 67 kilodaltons in quantities ranging from low levels in normal fibroblasts to 10-fold-higher levels in Epstein-Barr virus-immortalized and Burkitt's lymphoma cell lines, to 20- to 60-fold-higher levels in cell lines having amplified c-myc. The p64 and p67 proteins were found to be highly related by partial V8 proteolytic mapping, and both were demonstrated to be encoded by the c-myc oncogene, using hybrid-selected translation of myc-specific RNA. In addition, the p64 protein was specifically precipitated from cells transfected with a translocated c-myc gene. Both p64 and p67 were found to be nuclear phosphoproteins with extremely short half-lives. In tumor cell lines having alterations at the c-myc locus due to amplification or translocation, we observed a significant change in the expression of p64 relative to p67 when compared with normal or Epstein-Bar virus-immortalized cells.     

DNase I hypersensitivity in the gamma globin gene locus of K562 cells.

We have probed the chromatin conformation of the G gamma-A gamma-delta-beta globin gene locus of K562 cells, a human hematopoietic cell line, with the enzyme pancreatic DNAse I. This enzyme preferentially digests genes in an active configuration. We have found that in K562 cells, which produce embryonic and fetal but not adult hemoglobins, both the active gamma and inactive beta genes are DNAse I sensitive. However, only the active gamma genes have DNAse I hypersensitive regions. The hypersensitive regions have been mapped to an area approximately 100 base pairs 5' to the G gamma and A gamma genes.     

In vivo stimulation by antitumor drugs of the topoisomerase II induced cleavage sites in c-myc protooncogene

Several antitumor drugs including DNA intercalative and non intercalative agents induce in vitro and in vivo double-stranded DNA breaks by stabilization of a topoisomerase II-DNA complex. In order to locate cleavage sites in an actively transcribed oncogene, N417 cells, originating from a human small cell lung carcinoma and containing 45-50 copies of c-myc oncogene, were treated with mAMSA, 9 hydroxyellipticine and VM 26. The presence of DNA lesions in c-myc was investigated by Southern blot hybridization with a human c-myc probe. In addition to normal bands, DNA patterns of drug treated-cells revealed the presence of new bands most likely corresponding to topoisomerase II-mediated cleavage as these bands were not found in untreated control DNA and in DNA treated with oAMSA, a biologically inactive stereoisomer of mAMSA. Major cleavage sites induced by drugs in the N417 cell c-myc locus were located in the 5' end of the c-myc exon 1 closely to some DNAse I hypersensitive sites which are assumed to reflect an activity of the gene. Therefore our data suggest that TopoII-mediated drug activity correlates with gene activity.     

Elimination of extrachromosomal c-myc genes by hydroxyurea induces apoptosis

Hydroxyurea (HU) increases extrachromosomal DNA elimination in tumor cell lines. The c-myc oncogene is one of the many relevant amplified genes contained within the extrachromosomal DNA compartment. Spontaneous loss of amplified copies of c-myc induces terminal differentiation and apoptosis in the human HL-60 leukemia cell lines. In the present study, we evaluate HU's ability to induce apoptosis by eliminating extrachromosomally located c-myc oncogene in human tumor cell lines. The consequences of eliminating extrachromosomal DNA by HU were explored in two different cell lines using the TdT assay and acridine orange/ethidium bromide labeling. COLO 320 clone 3 and COLO 320 clone 21 cell lines contain the same number of amplified copies of c-myc oncogene, but located respectively on extrachromosomal DNA, and intrachromosomally in homogeneously staining regions. HU induced apoptosis in the COLO 320 clone 3 cell line by a time and concentration dependent mechanism but could not induce apoptosis in the COLO 320 clone 21 cell line. These results suggested that HU-induced apoptosis in COLO 320 cell lines depends on elimination of extrachromosomal amplified copies of the c-myc oncogene. The ability of HU to eliminate extrachromosomally amplified copies of the c-myc oncogene and to induce apoptosis should be considered when targeting malignancies with amplification of the c-myc oncogene in an extrachromosomal site.     

Oncogene amplification correlates with dense lymphocyte infiltration in human breast cancers: A role for hematopoietic growth factor release by tumor cells?

One hundred six primary breast cancer samples were analysed for c-erbB2, int-2, and c-myc gene amplification. Surgically confirmed nodal involvement was observed in 42%. Level of gene amplification was studied by Southern and/or slot blot techniques. Amplified c-erbB2 gene sequences were present in 21.5% of all samples. Int-2 was amplified in 13.1% and c-myc was amplified in 10.3%. In a non-parametric test (Kruskal-Wallis) a strong negative association was found between high levels of c-erbB2 amplification and absence of estrogen receptor (ER) (P = .0009) or progesterone receptor (PR) (P = .011) expression. No correlations were found between all or high levels of amplification of each oncogene separately or combined with T, N, grade, multifocality of tumor, or associated carcinoma in situ. There was a trend approaching statistical significance for patients with c-erbB2 amplifications to have positive lymph nodes at surgery (P = 0.09). A somewhat surprising finding however was a very strong association between oncogene amplification and dense lymphocyte infiltration of the tumor (P = .05). This correlation is even stronger when only high levels of amplification are considered, either for each oncogene separately (P = .0048) or in combination (P = .0007). We propose that malignant cell cytokine production may help explain this observation.     

Structure of a mouse erythroid 5-aminolevulinate synthase gene and mapping of erythroid-specific DNAse I hypersensitive sites.

The enzyme 5-aminolevulinate synthase (ALA-S) catalyzes the first step in heme biosynthesis. In this study, the mouse erythroid gene has been cloned and analyzed in order to investigate the regulation of ALA-S expression during erythroid differentiation. The gene spans approximately kbp and consists of 11 exons and 10 introns. The first exon is 37 bp, non-coding, and followed by a 6kb intron. The mRNA capsite was mapped by primer extension and defines a promoter that contains no apparent TATA element. S1 nuclease analysis detects the presence at low levels of a 45 bp-deleted form of the ALA-S mRNA created by the use of an alternative splice site at the intron 2/exon 3 junction. Five DNAse I hypersensitive sites were detected in chromatin from uninduced and induced MEL cells. One site is at the promoter; the others are in the body of the gene. No significant differences were observed in the patterns or intensity of the hypersensitive sites in the uninduced and induced MEL cells, however, no sites in ALA-S were observed in NIH 3T3 cells or in deproteinized DNA. Thus, these sites are specific for erythroid chromatin but appear to be established at an earlier stage of differentiation than represented by the uninduced MEL cell.     

Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction

The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19. Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells. A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.