Age-associated changes in DNA methylation and mRNA level of the c-myc gene in spleen and liver of mice

Age-associated changes in DNA structure and mRNA level were studied for the c-myc gene in spleen and liver of mice at 2, 14 and 26 months of age. Neither amplification nor rearrangement of the gene was detected in either tissue at any age. However, a significant alteration was observed in the methylation profile. The profile of the gene and its vicinity was determined using various methylation-sensitive restriction enzymes. In both tissues, the gene had an unmethylated domain ranging from −2 kb upstream of the 5′ end of the first exon to the 3′ end of the first intron. It was flanked by partially methylated regions, where age-dependent changes as well as tissue specificity were observed. In the spleen, age-related hypomethylation was observed at both the 3′ and 5′ sides of the domain. In contrast, hypermethylation was found in the liver only at the 3′ side. The steady-state level of c-myc mRNA showed a drastic decrease in liver from youth to middle age, while splenic mRNA changed little. The correlation between the changes of mRNA and DNA methylation is discussed.     

Effects of energy restriction on age-associated changes of DNA methylation in mouse liver

DNA methylation is known to change with age in several mammalian species. Here we have examined the effect of dietary energy restriction on this age-associated change in liver DNA of C3H/SHN mice. The total 5-methyldeoxycytidine level in the genome decreased slightly soon after energy restriction started. The effect, however, diminished with time and no appreciable difference was detected at middle and old ages. The degree of methylation at the c-myc gene, on the other hand, was not affected by energy restriction at early periods, but the age-dependent alterations at later ages were repressed. This is a new finding to show that DNA methylation is one of the molecular indices of aging affected by energy restriction. It suggests an importance of DNA methylation in the aging process.     

c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets

We report here that the expression of virtually all proposed c-Myc target genes is unchanged in cells containing a homozygous null deletion of c-myc. Two noteworthy exceptions are the gene cad, which has reduced log phase expression and serum induction in c-myc null cells, and the growth arrest gene gadd45, which is derepressed by c-myc knockout. Thus, cad and gadd45 are the only proposed targets of c-Myc that may contribute to the dramatic slow growth phenotype of c-myc null cells. Our results demonstrate that a loss-of-function approach is critical for the evaluation of potential c-Myc target genes.     

Mammary carcinogenesis is preceded by altered epithelial cell turnover in transforming growth factor-alpha and c-myc transgenic mice

Identification of biomarkers that indicate an increased risk of breast cancer or that can be used as surrogates for evaluating treatment efficacy is paramount to successful disease prevention and intervention. An ideal biomarker would be identifiable before lesion development. To test the hypothesis that changes in cell turnover precede mammary carcinogenesis, we evaluated epithelial cell proliferation and apoptosis in mammary glands from transgenic mice engineered to develop mammary cancer due to expression in mammary epithelia of transforming growth factor α (TGF-α) or c-myc. In transgenic glands, before lesion development, epithelial cell turnover was enhanced overall compared with nontransgenic glands, indicating that aberrant cell turnover in normal epithelia may contribute to tumorigenesis. In addition, in tumor-containing glands, proliferation in normal epithelia was higher than in tumor-free transgenic glands, suggesting these cell populations influence one another. Finally, although c-myc glands displayed a uniformly high epithelial cell turnover regardless of age, cell turnover was reduced with aging in nontransgenic and TGF-α mice, indicating that some growth and death regulatory mechanisms remain intact in TGF-α epithelia. These observations support the evaluation of cell turnover as a biomarker of cancer risk and indicator of prevention/treatment efficacy in preclinical models and warrant validation in human breast cancer.     

Catecholamine-induced cardiac hypertrophy: significance of proto-oncogene expression

The effect of β- and α-adrenergic stimulation on cardiovascular function and development of cardiac hypertrophy was studied in rats by measuring the heart weight/body weight and cardiac RNA/DNA ratios. β-Receptor stimulation with isoproterenol over 3 days induced an increase in the biosynthesis of cardiac adenine nucleotides, myocardial protein synthesis, and the heart weight/body weight ratio. The isoproterenol-induced metabolic effects were prevented by simultaneous β-adrenergic blockade with propranolol. α-Adrenergic stimulation with norfenephrine for 3 days induced an increase in heart rate, total peripheral resistance, the myocardial RNA/DNA, and left ventricular weight/body weight ratio. The calcium antagonist verapamil prevented the hemodynamic changes but did not influence the development of cardiac hypertrophy. The α-adrenergic blocker prazosin reversed the norfenephrine-induced functional changes and prevented cardiac hypertrophy. Norepinephrine was infused into isolated perfused working rat hearts to elucidate some molecular biological changes that precede the development of cardiac hypertrophy. It increased transiently and sequentially the mRNA of c-fos and c-myc. This enhancement occurred at about the same time as that induced by elevation of pre- and afterload but was more pronounced. These findings were compared with those obtained in other studies assessing the effects of catecholamines on proto-oncogene expression. Combination of norepinephrine with pre- and afterload elevation induced the c-fos mRNA signal to appear earlier, to be more pronounced, and to persist for a longer period of time. Similar results were obtained in regard to the c-myc mRNA. These findings indicate that the combination of two hypertrophy-inducing stimuli which may cause a higher degree of cardiac hypertrophy in vivo induce an earlier, more pronounced, and longer lasting expression of the proto-oncogenes c-fos and c-myc.     

Human and mouse cellularmyc protooncogenes reside on chromosomes involved in numerical and structural aberrations in cancer 1

A molecular clone of viralmyc (v-myc, the oncogene of avian myelocytomatosis virus, MC29, detected homologous human, mouse, and Chinese hamster cellularmyc (c-myc sequences by Southern filter hybridization. A v-myc probe, containing sequences from the 3′ domain of the gene, hybridized to single human HindIII and mouse EcoRI genomic DNA fragments of the cellular myc genes whose segregation could be followed in interspecies somatic cell hybrids. Human c-myc segregated concordantly with the enzyme marker glutathione reductase and with a karyotypically normal chromosome 8. A rearrangement of human c-myc was observed in Burkitt's lymphoma cells possessing the t(8;14) translocation. These results suggest that human c-myc is located close to the breakpoint on chromosome 8 (q24) involved in the t(8;14) translocation. The mouse c-myc gene segregated concordantly with chromosome 15 in mouse-Chinese hamster cell hybrids. These gene assignments are noteworthy, as structural and numerical abnormalities of human chromosome 8 and mouse chromosome 15 are associated frequently with B-cell neoplasms.     

No correlation of c-myc overexpression and p53 mutations in liposarcomas

 Although it is well known that oncogenesis is a multistep process involving the activation of normal cellular genes to become oncogenes and/or the inactivation of tumor suppressor genes, this process has seldom been investigated in soft tissue tumours. We screened a group of 36 liposarcomas for genetic abnormalitis in the p53 tumour suppressor gene and c-myc oncogene. Altered c-myc gene expression was examined by differential RT-PCR assay. p53 Gene mutations in exons 4–8 were analysed by using PCR-SSCP analysis and direct sequencing. Elevated c-myc expression was found in 6 of 31 liposarcomas (19.4%). p53 Gene mutations were observed in 5 of 36 liposarcomas (13.9%). Both genetic alterations were associated with the histological subtype of liposarcomas. Whereas c-myc gene expression was a characteristic of myxoid/round cell liposarcomas, p53 gene mutations were found more frequently in pleomorphic variants. Liposarcomas of the well-differentiated subtype showed neither p53 gene mutations nor altered c-myc gene expression. Our results indicate that the c-myc oncogene and the p53 tumor suppressor gene do not seem to cooperate in the oncogenesis of liposarcomas. Received: 22 April 1998 / Accepted: 11 May 1998     

Analysis of c-myc DNA amplification in non-small cell lung carcinoma in comparison with small cell lung carcinoma using polymerase chain reaction

Previous studies of c-myc DNA amplification in lung cancer have focused primarily on analysis of small cell carcinoma or its tumor cell lines. There are few data about c-myc DNA amplification in histological types of lung cancer other than small cell carcinoma. Therefore the present study was conducted to investigate c-myc oncogene amplification in non-small cell lung carcinoma. We studied 46 lung tumor specimens for c-myc DNA amplification (15 adenocarcinomas, 15 squamous cell carcinomas, 6 large cell carcinomas, and 10 small cell carcinomas). Polymerase chain reaction, digoxigenin DNA labeling, and elecrophoresis were utilized to investigate the c-myc copy number in the lung tumor specimens. The c-myc copy number of non-small cell carcinoma ranged from 1.5 to more than 20.0 in adenocarcinoma and squamous cell carcinoma, and from 6.0 to 12.0 in large cell carcinoma. That of small cell carcinoma ranged from 1.8 to 12.0. The c-myc copy number of non-small cell carcinoma was significantly higher that than of small cell carcinoma (Wilcoxon rank sum test, Z=2.06, P=0.040). However, the differences in c-myc copy number among these four histological types were not statistically significant. Amplification of c-myc (more than 4 copies) was observed not only in small cell carcinoma but also in non-small cell carcinoma at similarly high frequency (12/15 in adenocarcinoma and squamous cell carcinoma, 6/6 in large cell carcinoma, and 9/10 in small cell carcinoma). Received: 17 October 2000 / Accepted: 7 May 2001     

c-myc GENE ABNORMALITIES IN MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT) LYMPHOMAS

c-myc gene abnormalities associated with lymphomagenesis, including rearrangements and mutations in the regulatory region between exon I and intron I, have been studied in 54 MALT lymphomas (43 low and 11 high grade) and 36 nodal lymphomas (27 low and 9 high grade). By Southern blot analysis, none of the 54 MALT lymphomas but 2 of 36 nodal lymphomas had c-myc gene rearrangements. Defined tumour cell populations from all MALT lymphoma cases were isolated by microdissection from frozen tissue sections and analysed by polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) and direct sequencing for somatic mutations in the exon I/intron I region of the gene. Point mutations in this region were identified in nine cases of MALT lymphoma (7/43=16·2 per cent of low grade; 2/11=18·1 per cent of high grade). These mutations were located at either the exon I/intron I border of myc intron factor (MIF) binding sites, which are critical in the negative regulation of c-myc expression. Of the nodal lymphomas, only the two cases (5·6 per cent) with c-myc gene rearrangement showed scattered or clustered mutations. These results suggest that c-myc mutations in MALT lymphomas are unlikely to be associated with chromosome translocation, which is the main cause of somatic mutations observed in other types of lymphoma. The mutations involving the c-myc regulatory regions may play a pathogenetic role in at least a proportion of MALT lymphomas. © 1997 by John Wiley & Sons, Ltd.     

Expression of the c-myc and Ca-ATPase genes in cardiac muscle during adaptation to repeated stress

In the course of adaptation to repeated stress, the expression of the proto-oncogene c-myc found to increase much more rapidly than that of the Ca-ATPase gene. It is suggested that an increase in the level of c-myc expression may activate the structural Ca-ATPase gene and possibly also the heat-shock proteins. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 2, pp. 124–126, February, 1994     

Messenger RNA 3′-end formation of a DNA fragment from the human c-myc 3′-end region in Saccharomyces cerevisiae

We have tested the functioning of the human c-myc polyadenylation signal in Saccharomyces cerevisiae. A DNA fragment containing the two AATAAA polyadenylation signals of the c-myc gene was inserted into a plasmid designed for the in-vivo testing of polyadenylation signals in yeast. The c-myc fragment had a partial capacity for directing mRNA 3-end formation in yeast. The 3-endpoints were 50–100 bp distant from the mRNA 3-ends mapped in humans. This human DNA fragment is therefore unspecifically functional in yeast, indicating that other sequence elements than the human polyadenylation signal, AATAAA, are necessary for 3-end formation.     

Analysis of c-fos,c-jun,c-erbB1, c-erbB2 and c-myc in primary lung carcinomas and their lymph node metastases

The purpose of this study was to identify possible alterations in proto-oncogenes (c-fos, c-jun, c-erbB1, c-erbB2 and c-myc) at the protein level in primary lung carcinomas and simultaneous metastatic lymph nodes of 21 patients. The analysis showed that proteins of c-jun and c-myc were expressed in a significantly higher frequency in metastases than in primary lung tumors. Gross differences were not found between primary tumors and metastatic tumors with regard to the expression of c-erbB1, c-erbB2 and c-fos. The finding of cases with a higher expression of c-jun and c-myc in lymph nodes suggests that metastatic capability may be higher in certain cell populations.     

Expression of c-myc protein is related to cell proliferation and expression of growth factor receptors in transitional cell bladder cancer

Archival biopsy specimens from transitional cell bladder tumours (n=185) were analysed immunohistochemically for expression of c-myc protein. The results were compared with compared with histopathological and clinical parameters and survival. Forty-three per cent of the tumours were negative for c-myc protein and weak, moderate, or strong cytoplasmic expression was found in 34, 14, and 9 per cent of cases, respectively. Nuclear positivity for c-myc protein was detected in 35 per cent of tumours and nuclear opositivity was related to overexpression of c-erb B-2 (P=0.01) and a high proportion of nuclei were also positive for p53 oncoprotein (p<0.05). Cytoplasmic expression of c-myc protein was related to histological grade (P=0.005), papillary status (P=0.007), the S-phase fraction (P=0.008), the mitotic index (P=0.021), overexpression of epidermal growth factor receptor (P=0.045), and c-erb B-2 (P=0.17). Expression of c-myc protein was not significantly related to the progression of tumours and it had no prognostic value in survival analysis. Independent predictors were the T-category (P<0.001), papillary status. (P=0.001), and S-phase fraction (P=0.061). The results show that while c-myc gene product participates in growth regulation of human bladder cancer cells, it has no independent prognostic significance.     

Role of c-myc and CD45 in spontaneous and anti-receptor-induced apoptosis in adult murine B cells

Although adult murine B cells can be stimulated to proliferateby IgM receptor cross-linking, we and others have shown thatthese cells will undergo apoptosis in vitro in a dose-, time-and temperature-dependent manner with polyclonal but not monoclonalantl-lgM. To test the role of c-myc and cell cycle progressionin B cell apoptosis, we examined normal, Sp6 antl-TNP Ig andEµ-myc transgenic splenocytes for receptor-mediated apoptosisin vitro. In normal mice, both spontaneous and anti-lgM-lnducedprogrammed cell death were specifically blocked by antisenseoligodeoxynucleotides for the c-myc proto-oncogene, whereasnonsense myc oligonucleotides and irrelevant oligonucleotideshad only a minor effect. Similarly, TNP-dextran-induced apoptosisin Sp6 antl-TNP transgenics was inhibited by antisense c-myc.This effect was not due to the mitogenic effects of unmethylatedCpG-contalnlng sequences because ones lacking this motif, aswell as methylated oligonucleotides containing this motif, preventedapoptosis, and mitogenic doses of lipopolysaccharlde failedto Inhibit anti-lgM-driven cell death. Importantly, antisensec-myc also prevented the anti-lgM-lnduced increase in myc proteinspecies. Moreover, spontaneous apoptosis in vitro was exaggeratedin Eµ-myc transgenic B cells. To examine the role of CD45in anti-lgM-induced apoptosis, we treated spleen cells fromCD45 knockout mice, which do not proliferate with anti-IgM,and found that B cells from these underwent apoptosis normallydespite the lack of entry into S. These data suggest that anti-IgMdriven apoptosis does not require CD45. Rather, apoptosis maybe due to an overexpression of myc protein in the absence ofsignals which can drive B cells productively Into S, but thefailure to proliferate normally is insufficient for apoptosisto occur.