Structure of the polymorphic feline c-myc oncogene locus

Two feline c-myc DNA clones (CM-2 and CM-3), isolated from a cat DNA library, are structurally very similar. However, they differ at a SmaI site in exon III which is present only in CM-2. In the outbred feline population, cats heterozygous for this site or homozygous for the CM-3-type gene have been observed. The results provide a physical map of the feline c-myc locus, and define hitherto unidentified alleles of this gene.     

Analysis of the structure and expression of the c-myc oncogene in cervical tumor and in cervical tumor-derived cell lines

The structure of the c-myc oncogene in 17 cervical tumors and patient-matched nontumor tissues from Chinese patients residing in Taiwan was analysed. In contrast to recent reports on Mexican patients, none of the samples showed rearrangements and sequence amplification in the c-myc gene. The discrepancy may be explained by different carcinogenesis mechanisms being in operation in different geographic regions. Although no structural alterations in the c-myc gene were found in seven cervical carcinoma cell lines analysed, Northern blot analysis indicated different levels of c-myc gene expression which may be related to the presence of human papillomavirus (HPV) sequence in the cell and suggests a possible c-myc-hpv interaction in some stages of the transformation process.     

Analysis of the expression of the oncogene c-myc in human breast adenocarcinoma

The c-myc oncogene was characterized and its expression analyzed in 32 mammary adenocarcinomas and in 2 benign breast tumors from 34 untreated patients. Southern blot hybridization experiments have demonstrated the amplification of the oncogene (3 to 30 fold) in 3 carcinomas. The analysis of total RNA by Northern blot revealed the presence of a 2.4 kb c-myc RNA band. In 7 out of 10 carcinomas from patients with 3 or more than 3 lymph node metastases the level of c-myc expression evaluated by dot blot analysis was 4 to 14 fold greater than that of normal human tissues. In only 5 out of 22 carcinomas from patients without lymph node metastases or less than 3 invaded nodes the level of c-myc expression was also higher (4 to 10 fold). The level of c-myc expression was not significantly enhanced in the 2 benign tumors. It is suggested that the c-myc gene activation could be associated to a higher degree of malignancy of mammary carcinomas.     

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.     

Amplification of the expression of the c-myc oncogene in bronchial epidermoid carcinoma in man

Squamous cell lung carcinomas from 10 untreated patients were examined for the state of the oncogene c-myc. Blot hybridization experiments have demonstrated the amplification of the oncogene of about six fold in only one tumor. The oncogene amplification was not detected in normal tissues of patients. The analysis of RNA by Northern blot revealed the presence in the seven tumors examined of a 2.4 kb c-myc RNA band. The level of c-myc expression evaluated by dot blot analysis was 5 to 14 fold greater in tumors than that of histologically normal lung of the same patients.     

Requirement of lipocalin 2 for hematopoietic and solid tumor malignancies

Our published studies indicate that lipocalin 2 is a critical factor for invasion of hematopoietic tissues by chronic myelogenous leukemia cells. Lipocalin 2 is secreted by Bcr-Abl+ chronic myeloid leukemia cells causing apoptosis in normal hematopoietic cells but not in Bcr-Abl+ cells. Secreted lipocalin 2 forms a complex with metalloproteinase MMP-9, and is reported to stabilize its activity, which implies that lipocalin 2 may have a role in solid tumor formation. Our studies indicate that Bcr-Abl+ mouse marrow cells lacking the lipocalin 2 gene are not only defective for leukemia induction but also are defective in forming solid tumors compared to Bcr-Abl marrow cells that express lipocalin 2. Thus, our findings indicate that lipocalin 2 has two functions in tumor malignancies: one involving induction of apoptosis in normal hematopoietic cells and the other to facilitate solid tumor formation.     

Amplification and expression of a cellular oncogene (c-myc) in human gastric adenocarcinoma cells.

Three of 16 human gastric adenocarcinoma samples, maintained as solid tumors in nude mice, were found to carry amplified c-myc genes. In two samples with a high degree of c-myc DNA amplification (15- to 30-fold), double minute chromosomes were observed in karyotype analysis. The level of c-myc RNA was markedly elevated in a rapidly growing and poorly differentiated tumor, whereas it was only slightly elevated in a slowly growing and more differentiated tumor.     

Analysis of polyadenylation site usage of the c-myc oncogene.

The c-myc gene contains 2 well conserved polyadenylation (pA) sites. In all human and rat cell lines from various differentiation stages and tissue types the amount of mRNA terminating at the second pA site is 6-fold higher than the amount ending at the upstream site. This is not due to a difference in stability of the two mRNA types and therefore must be due to preferential usage of the downstream site. The usage of the pA sites is not altered during growth factor induction of quiescent cells. We have not been able to detect differences in behavior between mRNAs ending at either pA site. Both types of mRNA are induced upon treatment of cells with cycloheximide. Furthermore, we have shown that the poly(A) tail of c-myc mRNA is lost during degradation of the messenger, as was described previously for c-myc mRNA in an in vitro system. The time required for the loss of the poly(A) tail is similar to the half-life of c-myc mRNA.     

Targeted expression of a conditional oncogene in hematopoietic cells of transgenic mice

We have produced two lines of transgenic mice in which the expression of temperature-sensitive SV-40 large T antigen is targeted to bone marrow megakaryocytes via the platelet factor 4 (PF4) tissue-specific promoter. The progeny of these transgenic mice were observed for about 3 mo, and no malignancies were detected over this period of time. The offspring of these transgenic mice, 6- to 12-wk of age, served as a source of bone marrow cells, which upon in vitro cultivation at the permissive temperature yielded immortalized cell lines (MegT). At the permissive temperature, MegT cells exhibit the characteristics of early 2N and 4N megakaryocytes which include the presence of specific gene products such as PF4, glycoprotein IIb, acetylcholinesterase, and CD45 as well as the absence of molecular markers of other cell lineages such as the macrophage marker Mac-1, the T helper cell marker CD4, the mast cell marker IgE, the T cell marker CD2 or the erythroid cell marker alpha-globin. The inactivation of the oncogene by a shift of temperature from 34 degrees to 39.5 degrees C produces a reduction in the frequency of the 2N cells, in conjunction with the appearance of 8N and 16N cells, consisting of 27 and 3% of total cells, respectively. Thus, we have generated hematopoietic cell lines that are trapped in the early stages of megakaryocyte commitment, but able to undergo part of the normal program of terminal differentiation.     

UV damage and repair in the domain of the human c-myc oncogene

We have used a modification of the Southern hybridization method to analyze the removal of UV-induced pyrimidine cyclobutane dimers from the domain of the c-myc oncogene. The study was performed in human COLO320HSR cells, which exhibit a 30- to 40-fold amplification of c-myc that is maintained in a marker chromosome as a homogeneously staining region. Intron 2 and the region upstream from the gene showed better dimer removal than intron 1 or the region downstream from the c-myc gene. Regions showing less repair coincide with regions that are hotspots for mutations and chromosome translocations. Therefore, it is proposed that the inefficiency of DNA repair may play an important role in the origin of c-myc rearrangements.     

The effects of hydrocortisone on c-fos, c-myc and c-ras oncogene expression in IMR-90 fibroblasts

The effects of hydrocortisone on oncogene expression in human IMR-90 fibroblasts was analyzed by Northern blotting of total RNA. In synchronized fibroblasts stimulated with serum alone, there were two time periods of increased c-fos expression during the G1 phase of the cell cycle. There was no significant difference between cells treated with serum plus hydrocortisone, and cells treated with serum alone with respect to c-fos expression. Quiescent cells showed no change in c-fos expression during the G1 phase of the cell cycle. Three peaks of c-fos expression occur when cells are treated with hydrocortisone alone, but hydrocortisone in the absence of serum is insufficient to initiate DNA synthesis. Hydrocortisone has no effect on c-myc or c-Ha-ras expression in the presence or absence of serum in synchronized fibroblasts. Therefore, the control of mRNA production of the nuclear oncogenes c-fos and c-myc, and the cytoplasmic oncogene c-ras are independent and hydrocortisone may enhance DNA synthesis by increasing c-fos expression.     

Suppression of c-myc oncogene expression by a polyamine-complexed triplex forming oligonucleotide in MCF-7 breast cancer cells.

Polyamines are excellent stabilizers of triplex DNA. Recent studies in our laboratory revealed a remarkable structural specificity of polyamines in the induction and stabilization of triplex DNA. 1,3-Diaminopropane (DAP) showed optimum efficacy amongst a series of synthetic diamines in stabilizing triplex DNA. To utilize the potential of this finding in developing an anti-gene strategy for breast cancer, we treated MCF-7 cells with a 37mer oligonucleotide to form triplex DNA in the up-stream regulatory region of the c-myc oncogene in the presence of DAP. As individual agents, the oligonucleotide and DAP did not downregulate c-myc mRNA in the presence of estradiol. Complexation of the oligonucleotide with 2 mM DAP reduced c-myc mRNA signal by 65% at 10 microM oligonucleotide concentration. In contrast, a control oligonucleotide had no significant effect on c-myc mRNA. The expression of c-fos oncogene was not significantly altered by the triplex forming oligonucleotide (TFO). DAP was internalized within 1 h of treatment; however, it had no significant effect on the level of natural polyamines. These data indicate that selective utilization of synthetic polyamines and TFOs might be an important strategy to develop anti-gene-based therapeutic modalities for breast cancer.     

Toxicity and toxicokinetics of a phosphorothioate oligonucleotide against the c-myc oncogene in cynomolgus monkeys.

A 2-week toxicity and toxicokinetic study of a 15-mer phosphorothioate oligonucleotide, INX-3280, against the c-myc oncogene was performed in cynomolgus monkeys. As this oligonucleotide readily adopts an aggregate structure, a quadruplex, which may be associated with adverse physiologic effects, this study was performed using INX-3280 that had been converted to its monomeric form. Animals received intravenous (i.v.) infusions of monomeric INX-3280 three times per week for 2 weeks at doses of 3 or 15 mg/kg per administration. The monkeys were examined for clinical signs: changes in hematology, serum chemistry, coagulation, and urinalysis parameters; complement activation; macroscopic findings at necropsy; and histopathologic alterations. In addition, the toxicokinetics of INX-3280 were evaluated, using a validated HPLC assay, after the first and last (sixth) doses. No treatment-related clinical signs of any adverse effects were observed, and there were no test article-related changes in hematology, serum chemistry, or complement activation parameters. The only alteration in clinical pathology parameters was a minor (30%) prolongation of the activated partial thromboplastin time (aPTT), reflecting slight inhibition of the intrinsic coagulation pathway, which was less than that reported with other oligonucleotides given at similar doses. Treatment-related histopathologic alterations consisted of characteristic accumulation of basophilic material in the cytoplasm of tubular epithelial cells in the kidney, resident macrophages in the lymph nodes, and Kupffer cells in the liver. These changes were graded as minimal in all cases. The basophilic material is believed to reflect accumulation of the oligonucleotide or metabolites or both. The pharmacokinetic parameters of INX-3280 were identical on the first and sixth administrations and were similar to those reported for other phosphorothioate oligonucleotides. Maximum concentration (Cmax) values for INX-3280 (101-119 microg/ml) were in excess of the threshold plasma concentrations reported to trigger complement activation by phosphorothioate oligonucleotides. It is concluded that the safety profile of monomeric INX-3280 in cynomolgus monkeys is quite favorable relative to the known effects of other phosphorothioate oligonucleotides, particularly with respect to the blood level-related toxicities of this class of compounds, including complement activation and inhibition of coagulation. This study found no toxicities that were expected to be clinically significant.