Cellular MYCro economics: Balancing MYC function with MYC expression.

The expression levels of the MYC oncoprotein have long been recognized to be associated with the outputs of major cellular processes including proliferation, cell growth, apoptosis, differentiation, and metabolism. Therefore, to understand how MYC operates, it is important to define quantitatively the relationship between MYC input and expression output for its targets as well as the higher-order relationships between the expression levels of subnetwork components and the flow of information and materials through those networks. Two different views of MYC are considered, first as a molecular microeconomic manager orchestrating specific positive and negative responses at individual promoters in collaboration with other transcription and chromatin components, and second, as a macroeconomic czar imposing an overarching rule onto all active genes. In either case, c-myc promoter output requires multiple inputs and exploits diverse mechanisms to tune expression to the appropriate levels relative to the thresholds of expression that separate health and disease.

Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization.

BACKGROUND: It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH). METHODS: Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. RESULTS: The mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers. CONCLUSIONS: Thus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A-bomb radiation may affect the increased amount of CNA as a hallmark of GIN and, subsequently, be associated with a higher histologic grade in breast cancer found in A-bomb survivors.

Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair.

BACKGROUND: In eukaryotic cells, there are two sub-pathways of nucleotide excision repair (NER), the global genome (gg) NER and the transcription-coupled repair (TCR). TCR can preferentially remove the bulky DNA lesions located at the transcribed strand of a transcriptional active gene more rapidly than those at the untranscribed strand or overall genomic DNA. This strand-specific repair in a suitable restriction fragment is usually determined by alkaline gel electrophoresis followed by Southern blotting transfer and hybridization with an indirect end-labeled single-stranded probe. Here we describe a new method of TCR assay based on strand-specific-PCR (SS-PCR). Using this method, we have investigated the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related protein kinases (PIKK) family, in the TCR pathway of UV-induced DNA damage. RESULTS: Although depletion of DNA-PKcs sensitized HeLa cells to UV radiation, it did not affect the ggNER efficiency of UV-induced cyclobutane pyrimidine dimers (CPD) damage. We postulated that DNA-PKcs may involve in the TCR process. To test this hypothesis, we have firstly developed a novel method of TCR assay based on the strand-specific PCR technology with a set of smart primers, which allows the strand-specific amplification of a restricted gene fragment of UV radiation-damaged genomic DNA in mammalian cells. Using this new method, we confirmed that siRNA-mediated downregulation of Cockayne syndrome B resulted in a deficiency of TCR of the UV-damaged dihydrofolate reductase (DHFR) gene. In addition, DMSO-induced silencing of the c-myc gene led to a decreased TCR efficiency of UV radiation-damaged c-myc gene in HL60 cells. On the basis of the above methodology verification, we found that the depletion of DNA-PKcs mediated by siRNA significantly decreased the TCR capacity of repairing the UV-induced CPDs damage in DHFR gene in HeLa cells, indicating that DNA-PKcs may also be involved in the TCR pathway of DNA damage repair. By means of immunoprecipitation and MALDI-TOF-Mass spectrometric analysis, we have revealed the interaction of DNA-PKcs and cyclin T2, which is a subunit of the human transcription elongation factor (P-TEFb). While the P-TEFb complex can phosphorylate the serine 2 of the carboxyl-terminal domain (CTD) of RNA polymerase II and promote transcription elongation. CONCLUSION: A new method of TCR assay was developed based the strand-specific-PCR (SS-PCR). Our data suggest that DNA-PKcs plays a role in the TCR pathway of UV-damaged DNA. One possible mechanistic hypothesis is that DNA-PKcs may function through associating with CyclinT2/CDK9 (P-TEFb) to modulate the activity of RNA Pol II, which has already been identified as a key molecule recognizing and initializing TCR.

Characterization of a radiation-induced stress response communicated in vivo between zebrafish.

Radiation-induced communication of stress signals between rainbow trout (Oncorhynchus mykiss W) have recently been described by this group and linked to the bystander effect. This paper addresses the question of whether another totally unrelated fish species (Danio rerio L) can demonstrate the effect and also looks at attenuation of both the bystander signal, from irradiated fish, and the bystander effect, in naive fish. The data show that zebrafish produce bystander signals, and that, as with rainbow trout these can affect naïve (i.e., non-irradiated) fish placed in water with X-rayed fish or in water previously occupied by X-rayed fish. Skin explants from directly X-rayed fish still reduce HPV-G reporter cell growth 6 h after X-ray, but the bystander signal to naïve fish is lost. Twelve h after X-ray the signal is lost in X-rayed fish. The bystander effect is also attenuated if induction was by placing naïve fish in water which previously held the X-rayed fish. However, the effect is retained if induction was by placing X-rayed and naïve fish together. This suggests the signal is not retained by water for long periods of time. Individual fish data reveal unique responses by bystander fish which could indicate varying levels of sensitivity to signal strength among individuals.

Defective expression of Notch1 and Notch2 in connection to alterations of c-Myc and Ikaros in gamma-radiation-induced mouse thymic lymphomas.

Gamma-radiation-induced thymic lymphomas constitute a heterogeneous group of T-cell lymphomas. Some tumour suppressor genes and oncogenes have been shown to be defective in a fraction of such lymphomas, yet a considerable number of these remain elusive in terms of gene alterations. In the present work we present evidence that gamma-radiation-induced thymic lymphomas in (C57BL/6 J x BALB/c) F1 hybrid mice often exhibit increased levels of Notch1 expression, but, contrary to what was expected, they also exhibit a clearly reduced Notch2 mRNA expression, suggesting a cooperative antagonism of these genes. These results represent the first reported instance for the involvement of Notch2 inactivation in the development of thymic primary tumours while confirming the role of Notch1 as an activated oncogene. Additional analyses revealed that c-Myc over-expression and partial inactivation of Znfn1a1/Ikaros appear to be relevant events some how coupled to alterations in Notch genes inducing these kinds of tumours.

Radiation therapy depletes extrachromosomally amplified drug resistance genes and oncogenes from tumor cells via micronuclear capture of episomes and double minute chromosomes.

PURPOSE: To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines. MATERIALS AND METHODS: The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR1 drug resistance genes and MYCC oncogenes, were irradiated with 2 Gy fractions up to a total dose of 28 Gy. To track the fate of extrachromosomally amplified genes, cells surviving radiation therapy and unirradiated control cells were analyzed by fluorescent in situ hybridization of chromosomes using MDR1 and MYCC-specific cosmid DNA probes. In addition, total DNA and protein isolated from irradiated and control cells was subjected to Southern and Western blotting procedures, respectively, to determine amplified gene copy number and protein expression levels. Dose-response assays to follow loss of function of the MDR1 gene from KB-C1 cells were also performed. RESULTS: A significant reduction in extrachromosomal DNA, amplified gene copy number, and expression was detected in surviving cells after relatively low doses of radiation. Entrapment of extrachromosomal DNA into micronuclei was a consistent feature of irradiated cells. CONCLUSIONS: Clinically relevant doses of radiation can deplete extrachromosomal DNA in viable human malignant cells and alter their phenotype. Depletion of extrachromosomally amplified genes from tumor cells occurs via entrapment in radiation-induced micronuclei.

Elliptically polarized magnetic fields do not alter immediate early response genes expression levels in human glioblastoma cells.

Expression of immediate early response genes such as c-fos, c-jun, and c-myc in response to 1-500 microT resultant (r) 60 Hz elliptically polarized (EP) magnetic fields (MFs), typical of environmental MFs polarization under overhead power lines, was analyzed in both at transcriptional and translational levels using human glioblastoma (T98G) cells. Pseudo synchronized T98G cells at G1 phase were exposed to EP-MFs (1, 20, 100, and 500 microTr) for up to 3 h, but produced no statistical difference (P>0.05) in the levels of expression ratio at both the transcriptional and translational levels at 30 min for c-fos and c-jun and at 180 min for c-myc after serum stimulation. In addition, exposure of T98G cells to linearly (vertical and horizontal) and/or circularly polarized MFs (500 microTr) produced no significant change (P>0.05) in the expression ratio at both transcriptional and post-transcriptional levels. Thus, there was no evidence that linearly or rotating polarized MFs enhanced early response gene expression in these studies. These results suggest that environmental MFs at 1-500 microT flux density are unlikely to induce carcinogenesis through a mechanism involving altered expression of the immediate early response genes.

Evaluation of in vitro effects of 50 and 60 Hz magnetic fields in regional EMF exposure facilities.

A weak association between magnetic-field exposure and increased incidences of cancer has been reported. While alterations in cellular processes after in vitro magnetic-field exposures have also been reported to provide plausibility for this association, other laboratories have been unable to repeat the findings. As part of an accelerated electric- and magnetic-field (EMF) research program, the National Institute of Environmental Health Sciences with the Department of Energy identified the replication of the published positive effects as a priority. Regional EMF exposure facilities were established to investigate major in vitro effects from the literature. These included effects on gene expression, intracellular calcium, colony growth in soft agar, and ornithine decarboxylase activity. The laboratories that first reported these effects provided experimental protocols, cell lines, and other relevant experiment details. Regional facility studies included sham/sham exposures (no applied field in either chamber) and were done in a blinded fashion to minimize investigator bias. In nearly all experiments, no effects of magnetic-field exposure were found. The effort provided insight into dealing with the difficulty of replication of subtle effects in complex biological systems. Experimental techniques provided some clues for the differences in experimental results between the regional facility and the original investigator. Studies of subtle effects require extraordinary efforts to confirm that the effect can be attributed to the applied exposure.

A dose response for radiation-induced intrachromosomal DNA rearrangements detected by inverse polymerase chain reaction.

X-ray-induced intrachromosomal DNA rearrangements were detected in the 5' region of the MYC gene of cells of the human bladder carcinoma cell line, EJ-30, by using PCR with inverted primers. When the cells were allowed to repair/misrepair for 6 or 23 h after irradiation, the frequency of rearrangements increased with dose from (0.7 +/- 0.4) x 10(-5) per copy of MYC for unirradiated cells to (3.2 +/- 0.7) x 10(-5) after 30 Gy, (5.4 +/- 1.2) x 10(-5) after 70 Gy, and (5.9 +/- 1.0) x 10(-5) after 100 Gy. No significant difference was observed between 6 and 23 h of repair. Sequences obtained from the products suggest that there was no homology between the two sequences involved in the recombination event and that there was no clustering of breakpoints. The procedure is relatively simple, requiring only one digestion with a rare-cutting restriction enzyme prior to PCR amplification of the DNA purified from irradiated cells. The site of enzyme digestion is located between a pair of primer sites 120 bp apart for which the primers face in opposite directions. If no intrachromosomal rearrangement has occurred, no PCR product would be obtained. However, if an intrachromosomal rearrangement has occurred between two regions located on either side of the primer sites, an episome or duplication event would result if the rearrangement had occurred either within the same chromatid or between two sister chromatids, respectively. Digestion between the primers would linearize an episome or release a linear molecule containing the duplicated primer sites from a larger molecule. After both types of rearrangement events, the primers would be facing each other and would be located on either end of the linear molecule; and if they are less than approximately 5 kb apart, PCR amplification should result in a product. This procedure is relatively simple and rapid and does not require any cell division after irradiation or phenotypic selection of mutants. Also, quantification is based on the number of PCR products detected in a known amount of DNA, and not on a precise determination of the amount of PCR amplification that has occurred. Thus the inverse PCR procedure has the potential ofbeing used as an assay to detect variations in radiation-induced frequencies of DNA rearrangements.

Gene expression in human breast epithelial cells exposed to 60 Hz magnetic fields.

Epidemiology suggests a possible relationship between exposure to power frequency magnetic fields (EMF) and breast cancer. One mechanism through which EMF could stimulate breast cancer induction is via altered expression of oncogenes and/or tumor suppressor genes that regulate normal and neoplastic growth. To evaluate the hypothesis that EMF action in the breast is mediated by alterations in gene expression, transcript levels of c-myc and a battery of other cancer-associated genes were quantitated in human breast epithelial cells exposed to pure, linearly polarized 60 Hz EMF with low harmonic distortion. HBL-100 cells and normal (non-transformed) human mammary epithelial cells were exposed to EMF flux densities of 0.1, 1.0 and 10.0 Gauss (G) for periods ranging from 20 min to 24 h; concurrent sham controls were exposed to ambient fields (<0.001 G) only. Gene expression was quantitated using ribonuclease protection assays. EMF exposure had no statistically significant effect on basal levels of c-myc transcripts in either human breast cell model, and had no effect on alterations in c-myc expression induced by 12-O-tetradecanoylphorbol-13-acetate. Transcript levels of c-erbB-2, p53, p21, GADD45, bax, bcl-x, mcl-1, and c-fos were also unaffected by EMF exposure. These results suggest that EMF is unlikely to influence breast cancer induction through a mechanism involving altered expression of these genes.

Proto-oncogene mRNA levels and activities of multiple transcription factors in C3H 10T 1/2 murine embryonic fibroblasts exposed to 835.62 and 847.74 MHz cellular phone communication frequency radiation.

This study was designed to determine whether two differently modulated radiofrequencies of the type generally used in cellular phone communications could elicit a general stress response in a biological system. The two modulations and frequencies studied were a frequency-modulated continuous wave (FMCW) with a carrier frequency of 835.62 MHz and a code division multiple-access (CDMA) modulation centered on 847.74 MHz. Changes in proto-oncogene expression, determined by measuring Fos, Jun, and Myc mRNA levels as well as by the DNA-binding activity of the AP1, AP2 and NF-kappaB transcription factors, were used as indicators of a general stress response. The effect of radiofrequency exposure on proto-oncogene expression was assessed (1) in exponentially growing C3H 10T 1/2 mouse embryo fibroblasts during their transition to plateau phase and (2) during transition of serum-deprived cells to the proliferation cycle after serum stimulation. Exposure of serum-deprived cells to 835.62 MHz FMCW or 847.74 MHz CDMA microwaves (at an average specific absorption rate, SAR, of 0.6 W/kg) did not significantly change the kinetics of proto-oncogene expression after serum stimulation. Similarly, these exposures did not affect either the Jun and Myc mRNA levels or the DNA-binding activity of AP1, AP2 and NF-kappaB in exponential cells during transit to plateau-phase growth. Therefore, these results suggest that the radiofrequency exposure is unlikely to elicit a general stress response in cells of this cell line under these conditions. However, statistically significant increases (approximately 2-fold, P = 0.001) in Fos mRNA levels were detected in exponential cells in transit to the plateau phase and in plateau-phase cells exposed to 835.62 MHz FMCW microwaves. For 847.74 MHz CDMA exposure, the increase was 1.4-fold (P = 0.04). This increase in Fos expression suggests that expression of specific genes could be affected by radiofrequency exposure.

Detection and sequencing of ionizing radiation-induced DNA rearrangements using the inverse polymerase chain reaction.

PURPOSE: To develop a procedure, using the inverse polymerase chain reaction, to detect and sequence ionizing radiation-induced DNA rearrangements without prior phenotypic selection of mutant cells. METHOD: Normal human fibroblast cells (IMR-90) were given 30Gy of gamma-irradiation and then incubated at 37 degrees C for 23h to allow DNA repair. Rearrangements of the sequence 5' to the c-myc gene were examined by amplifying the region using inverse PCR followed by DNA sequencing. RESULTS: Approximately fivefold more PCR products were amplified from the DNA of cells given 30 Gy of gamma-irradiation and allowed 23 h for repair than were obtained from cells that were either unirradiated or were irradiated and then lysed immediately. PCR products from seven putative radiation-induced DNA rearrangements were sequenced. Of these products, one contained an unidentified sequence (a possible inter-chromosomal rearrangement) whilst the other products appeared to derive from episomes or duplication events (possible intra-chromosomal rearrangements). The sequencing data suggested that the sites of DNA rearrangement breakpoints were non-randomly distributed and possibly associated with topoisomerase I consensus cleavage sequences. There was a significant level of direct homology between the sequences flanking the breakpoints. CONCLUSIONS: The procedure developed was able to detect both inter- and intra-chromosomal rearrangements.

Ultraviolet B suppresses vitamin D receptor gene expression in keratinocytes.

Keratinocytes not only produce vitamin D3 in response to ultraviolet B light (UVB) and convert 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D) but also possess the vitamin D receptor (VDR) and respond to 1,25(OH)2D. We characterized the regulation of the expression of the VDR gene in primary human keratinocytes following UVB irradiation. We report a marked dose-dependent down-regulation of the VDR mRNA and protein within a few hours after irradiation. This occurs independently of de novo protein synthesis and is not due to a change in the half-life of the VDR mRNA. Interestingly, treatment of the cells with sodium salicylate, caffeic acid phenethyl ester and tosylphenylchloromethylketone inhibited this down-regulation. Our results strongly suggest the existence of a feedback mechanism in that UVB initiates vitamin D synthesis in keratinocytes and at the same time limits VDR abundance. They also provide a rational explanation for the reported lack of any additive effect between 1,25(OH)2D and UVB phototherapy in the treatment of psoriasis.

Myc-mediated transactivation of HSP70 expression following exposure to magnetic fields.

We investigated c-myc protein-binding sites on the HSP70 promoter as modulators of the induction of HSP70 gene expression in response to magnetic field stimulation (8microT at 60Hz) and whether the presence of c-myc protein potentiates transactivation of HSP70 expression. A 320 base pair region in the HSP70 promoter (+1 to -320) was analyzed. This region contains two c-myc-protein binding sites with consensus sequences located at -230 and -160 nucleotide positions (relative to the transcription initiation site) and overlapping with the region reported for the regulation of HSP70 gene expression by c-myc protein. This promoter region is upstream of other regulatory sequences, including the heat shock element (HSE), AP-2, and serum response element (SRE). Transfectants containing both c-myc protein-binding sites, HSP-MYC A and HSP-MYC B, and exposed to magnetic fields showed a 3.0-fold increase in expression of CAT activity as compared with sham-exposed control transfectants. Transfectants containing one c-myc binding site, HSP-MYC A, and exposed to magnetic fields showed a 2.3-fold increase in CAT expression. Transfectants in which both HSP-MYC A and HSP-MYC B binding sites were deleted showed no magnetic field sensitivity; values were virtually identical with sham-exposed controls. If the c-myc expression vector was not co-transfected with the constructs containing myc-binding sites, there was no difference in the expression of CAT activity between magnetically stimulated and sham-exposed controls, although both responded to heat shock. These data suggest that endogenous elevated levels of myc protein contribute to the induction of HSP70 in response to magnetic field stimulation.

Effect of ionizing radiation on wild-type and mutant mouse leukemia L1210 cells.

Wild-type (WT) mouse leukemia L1210 cells express steady-state levels of p53 mRNA and protein. However, the p53 expressed by the wild-type L1210 cells was found to be a mutant form of p53 (relative to normal mouse fibroblast p53 sequence) having a point mutation in the DNA binding domain of p53. A deoxyadenosine-resistant L1210 cell line (Y8) derived from the parental WT cells had previously been shown to lack the expression of p53 but to respond to cycloheximide (CHX) treatment by superinduction of p53 mRNA. The mRNA for p53 induced by CHX had the same sequence as the p53 from normal mouse fibroblasts. Although the Y8 cells had no constitutive levels of p53 mRNA or protein, the Y8 cells expressed constitutive levels of WAF1 mRNA and protein. Gadd45 mRNA was also present in the Y8 cells. Subjecting the WT or Y8 cells to ionizing radiation did not result in a G0/G1 cell cycle block; the cells blocked in G2/M. The Y8 cells were much more sensitive to the irradiation treatment than the WT cells, resulting in marked increases in apoptosis in the Y8 cells. Although radiation treatment induced p53 mRNA, but no p53 protein, in the Y8 cells, WAF1 mRNA was induced in the Y8 cells. These data indicate that there are p53-independent pathway(s) that may still involve WAF1 and Gadd45 with respect to cell cycle control and apoptosis.

Influence of ionizing radiation on proliferation, c-myc expression and the induction of apoptotic cell death in two breast tumour cell lines differing in p53 status.

PURPOSE: To determine the capacity of ionizing radiation to inhibit proliferation, to suppress c-myc expression and to induce apoptotic cell death in the p53 wild-type MCF-7 cell line and the p53 mutated MDA-MB231 cell line. MATERIALS AND METHODS: Growth inhibition and cell killing were determined by cell number and trypan blue exclusion. Apoptosis was assessed through cell morphology and fluorescent end-labelling. c-myc expression was monitored by Northern blotting. RESULTS: Inhibition of cell proliferation by ionizing radiation was similar in both cell lines. MDA-MB231 cells accumulated in G2 while MCF-7 cells accumulated in both the G1 and G2 phases of the cell cycle after irradiation. There was no evidence of apoptosis in either cell line. In MCF-7 cells, growth inhibition correlated closely with an early dose-dependent suppression of c-myc expression; in MDA-MB231 cells, there was no correspondence between growth inhibition and a transient, dose-independent reduction in c-myc message. CONCLUSIONS: These findings suggest that in the absence of classical apoptotic cell death, radiosensitivity is not predictably related to the p53 status of the cell. While both p53 and c-myc may be linked to the DNA damage response pathway, neither p53 nor c-myc are essential for growth arrest in response to ionizing radiation.

Gene expression in skin tumors induced in hairless mice by chronic exposure to ultraviolet B irradiation.

We investigated the expressions of c-Ha-ras, c-jun, c-fos, c-myc genes and p53 protein in the development of skin tumors induced by chronic exposure to UVB without a photosensitizer using hairless mice. When mice were exposed to UVB at a dose of 2 kJ/m2 three times a week, increased c-Ha-ras and c-myc transcripts were detected after only 5 weeks of exposure, while no tumor appeared on the exposed skin. The increase in gene expression continued until 25 weeks, when tumors, identified pathologically as mainly squamous cell carcinomas (SCC), developed in the dorsal skin. In these SCC, overexpression of c-fos mRNA was also observed along with the increases in c-Ha-ras and c-myc. A single dose of UVB (2 kJ/m2) applied to the backs of hairless mice transiently induced overexpression of the early event genes c-fos, c-jun and c-myc, but not c-Ha-ras, in the exposed area of skin. Accumulation of p53 protein was determined by Western blotting analysis or immunohistochemistry using monoclonal antibodies PAb 240 or 246, which recognize mutant or wild type, respectively. In the SCC, a mutant p53 protein accumulated in the cytoplasm and nucleus. After single-dose irradiation, the increased wild-type p53 protein was observed in the nuclei of epidermal cells. The present results suggest that overexpression of the c-fos, c-myc and c-Ha-ras genes, and the mutational changes in p53 protein might be associated with skin photocarcinogenesis. Moreover, overexpression of the c-Ha-ras and c-myc genes might be an early event in the development of UVB-induced skin tumors in mice.

Oncogenic changes in murine lymphoid tumors induced by in utero exposure to ionizing radiation.

We have investigated the oncogenic alterations in murine lymphomas induced by in utero exposure to gamma-radiation. The expression of the myc oncogene increased in 23% of the tumors. Alterations in the expression of the ras oncogenes and in the p53 tumor suppressor gene were not characteristic. The p53 gene was mutated in a low percentage of the tumors (12%). Ras mutations were not detected. Loss of heterozygosity (LOH) at the p53 locus was found in 30% of the tumors, and LOH at the mts tumor suppressor gene was detected in 23% of lymphomas. Multiple oncogenic changes were infrequent in the investigated tumors. There were no essential differences in the frequency of carcinogenic alterations in spontaneous and gamma-radiation-induced lymphomas.

Carcinogen-induced de novo methylation in c-myc exon I.

During the response of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylation occurred at the Hpa II site of c-myc exon I, which is located downstream of the P1 initiation site, as evidenced by the assays of Hpa II-PCR. The Hpa II spite of the 5' flanking region did not undergo methylation. UV-irradiation also led to methylation in exon I. The extent of methylation increased depending on the dose of MNNG and UV. The results suggested that methylation takes place in transcriptionally active c-myc responsible for carcinogens and is caused by mechanisms different from that of alkylation in a specific CpG site. Possible contribution of methylation to less repair found in c-myc is discussed.