The involvement of HER2 and p53 status in the regulation of telomerase in irradiated breast cancer cells.

Cancer cell characteristics may play a pivotal role in the response to therapy by activating or deactivating different molecular pathways. In the present study, we investigated the implication of breast cancer cell features, such as HER2 and p53 in the activation of telomerase upon exposure to ionizing radiation. Telomerase is among the most important cancer biomarkers, conferring to tumor cells unlimited proliferative capacity, increased survival potential and resistance to several types of cellular stress. We investigated possible mechanisms regulating telomerase in six irradiated breast cancer cell lines (MCF-7, MCF-7/HER2, MDA-MB-231, SK-BR-3, BT-474 and HBL-100) differing in their HER2, p53 and ERalpha status. hTERT mRNA expression was evaluated by real-time PCR and telomerase activity by the TRAP assay. HER2, c-myc, p53 and p21 protein levels were evaluated by Western blotting. Silencing of hTERT and HER2 was achieved by small interfering RNA technology. Chromatin immunoprecipitation was used to evaluate H3 histone acetylation status, as well as myc/mad/max and p53 transcription factors interaction with the hTERT promoter. Our results showed for the first time, that only HER2-positive cells, independently of their p53 status, upregulated hTERT/telomerase, while knockdown of hTERT increased radio-sensitivity. Knockdown of HER2 also led to increased radio-sensitivity and downregulation of hTERT/telomerase. We also demonstrated that c-myc and mad1 regulate hTERT expression in all irradiated breast cancer cells. We conclude, for the first time, that HER2 phenotype upregulates hTERT through c-myc activation and confers radio-resistance to breast cancer cells.

c-MYC protein is degraded in response to UV irradiation.

The c-MYC proto-oncogene encodes a transcription factor that is critical for cell growth and proliferation. It is one of the genes frequently altered in cancer cells in which it exhibits constitutive activity. The half-life of c-MYC is very short in quiescent cells due to ubiquitin-mediated proteolysis. We report here the rapid and dose-dependent decline of c-MYC protein level after UV-irradiation in various human and rodent cells. This decline is due to a proteasomal degradation of c-MYC protein and does not require the binding sites for the FBW7 and SKP2 ubiquitin ligases. Together, our data exclude a prominent role for the stress-responsive kinase PAK2, for the major phosphoinositide 3-kinase related protein kinases ATR, ATM, DNA-PK and mTOR and for ERK, JNK and p38 mitogen activated protein kinases in this UV-induced degradation process. We propose that c-MYC degradation is part of the global cell response to UV-damage, complementary to the accumulation and activation of the p53 transcription factor. By contributing to the replication arrest after infliction of lesions to the genome, the induced degradation of c-MYC may be part of the safeguard mechanisms maintaining genome stability.

Apoptosis and other effects of radiation in normal human urothelial cells.

In this paper, an attempt is made to identify endpoints that might be of potential use in the quantification of radiation effects in human tissues. Irradiated cultures of cells that are not selected for clonogenic survival but are left in situ to grow after irradiation show a wide variety of morphological and biochemical abnormalities. These include nuclear fragmentation and other evidence of programmed cell death, but they also include a considerable amount of lysis, necrosis, and persistent abnormal growth and function, which are expressed in the progeny of irradiated cells. Induction of proteins associated with stress or shock responses, growth and cell cycle control, and control of apoptosis are also seen and may persist. The dose dependence of these various responses is documented, because it probably determines to a large extent the outcome of radiation exposure in terms of whether a cell dies, divides normally, or develops genomic instability, mutation, and ultimate carcinogenic progression of the progeny. Clearly, a cell that dies presents no further threat to the organism, nor does a fully repaired cell. Therefore, a major challenge facing radiation protection research is to define the population at risk of surviving with damage. The results show that there is a variation in response to radiation between different patient cultures that is detectable in an explant culture system of primary normal human urothelium. The growth pattern and protein expression postirradiation is consistent with apoptosis being a major determinant of low dose response to radiation. This form of death appears to be suppressed at higher doses and, in the majority of subjects, results in the presence of a highly abnormal population of cells, even though the population size is the same whether their progenitors were irradiated or not.

Variation in the expression of p53, c-myc, and bcl-2 oncoproteins in individual patient cultures of normal urothelium exposed to cobalt 60 gamma-rays and N-nitrosodiethanolamine.

The controls determining the initial response of cells to DNA damage probably determine whether a cancer will ultimately occur. Efficient repair or apoptosis represents extremes of control mechanisms. Misrepair can lead to fixation of damage. The changes in oncoprotein expression of three genes involved in the regulation of repair of DNA damage and postdamage proliferation of cells were measured in cultures of normal urothelium from 55 patients without any malignancy. The aim was to obtain information on interperson variation in response to carcinogens in the human population. The group included 10 pediatric patients < 2 years old. Two different carcinogenic agents, ionizing radiation and N-nitrosodiethanolamine, which represent widely different DNA-damaging pathways, were used. Both of these cause bladder cancer in humans. Cells from explanted tissue were examined after carcinogen exposure for levels of p53, c-myc, and bcl-2 proteins. Both carcinogens led to increased levels of cytoplasmic p53 protein expression, although there was significant interpatient variation. bcl-2 showed a very significant increase in expression after radiation exposure. c-myc was high and variable pre- and postexposure. Individual patient culture changes in the expression of the three oncoproteins did not correlate significantly with each other or with cell growth, suggesting that the controls are complex. Pediatric samples had lower mean control values of p53 and bcl-2 than did adult samples. This was due to the absence in this group of high controls seen in some adult cultures. The result suggest that an early breakdown in control mechanisms of growth arrest and apoptosis may occur in urothelium after carcinogen exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 50 Hz magnetic fields on C-myc transcript levels in nonsynchronized and synchronized human cells.

The effects of 50 Hz electromagnetic fields (EMFs) on the expression of the c-myc oncogene, known to be involved in normal cell proliferation and possibly also in tumor processes, were investigated in nonsynchronized human lymphoid cells immortalized by Epstein-Barr virus. Viral injury to such cells makes them a good model for exploring the possible cancer-promoting effects of 50 Hz magnetic fields. Parallel experiments were conducted on human HL60 leukemic cells. Cells were exposed to sinusoidal 50 Hz EMFs at 10 microT or 1 mT for 20 min, 1 h, 24 h, or 72 h. Exposure was performed either immediately after refeeding or 1.5 h after refeeding. C-myc transcript values were assessed by Northern blot analysis and normalized to those of the noninducible gene GaPDH. No statistically significant difference between the c-myc transcript levels of control and exposed cells was found in lymphoid or leukemic cells under our experimental conditions, either after short exposures of 20 min and 1 h or after longer exposures of 24 and 72 h. Other experiments were carried out with pseudosynchronized cells in an attempt to establish whether cells were especially sensitive to 50 Hz magnetic field exposure in any particular phase of the cell cycle. Accordingly, cells were pseudosynchronized in G0/G1 by serum deprivation and exposed for 20 min to a 50 Hz magnetic field, at 10 microT for lymphoid cells and 1 mT for HL60 cells. No significant difference was observed between the c-myc transcript levels of control and exposed cells for either of the synchronized cell types. These results for synchronized cells correlated with those for nonsynchronized cells.

[The repair of gamma-induced single-stranded breaks in the transcribed and nontranscribed DNA of HeLa cells]. / Reparatsiia gamma-indutsirovannykh odnonitevykh razryvov v transkribiruemoi i netranskribiruemoi DNK kletok HeLa.

The repair of gamma-ray-induced DNA single-strand breaks (SSB) in transcribed (Alu-enriched DNA, proto-oncogene c-myc) and non-transcribed (human satellite III) DNA of HeLa cells has been investigated. A special methodical approach has been developed. The method involved alkaline sucrose sedimentation followed by Southern hybridization in situ of 32P labelled plasmids (probes) containing sequences analysed with total DNA distributed through sucrose gradient fractions. The degree of the probes hybridization with cellular DNA was the criteria of the damage and that of DNA repair. The induction of DNA SSB after irradiation (100 Gy) in Alu-enriched DNA and c-myc gene was shown to be 1.3-1.4 time more often while than in satellite DNA, and 1.4 time lower compared to that in total DNA. The rate of DNA repair was different: the most part of lesions was eliminated in the first 10-20 minutes in all cases. For this time 60-67, 50-66, 35-50 and 45-50% DNA SSB were eliminated from transcribed DNA (c-myc, Alu), non-transcribed DNA (satellite III) and total DNA, respectively. Thus, the preferable (fast) repair of gamma-ray-induced DNA SSB takes place in transcribed DNA compared to that in non-transcribed DNA of HeLa cells.