Azemat J. Parsian

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.

Redox Factor-1 (Ref-1) Mediates the Activation of AP-1 in HeLa and NIH 3T3 Cells in Response to Heat Shock

The early response genes, c-Fos and c-Jun, are induced by environmental stress and are thought to modulate injury processes via the induction of AP-1-dependent target genes. AP-1 activation is thought to be regulated by changes in intracellular oxidation/reduction reactions involving the redox factor-1 (Ref-1) protein. In this study, NIH 3T3 and HeLa cells were used to determine whether heat shock induces the AP-1 transcription factor via signaling pathways involving Ref-1. Reverse transcriptase-polymerase chain reaction analysis and immunoblotting demonstrated that c-Fos and c-Jun were induced 2–10 h following heat shock, and this induction was accompanied by an increase in AP-1 DNA binding. Electrophoretic mobility shift assay extracts immunodepleted of Ref-1 protein demonstrated that the increase in AP-1 DNA-binding activity following heating was dependent upon the presence of Ref-1 and that Ref-1 regulates inducible, but not basal, AP-1 DNA-binding activity. This was confirmed by the restoration of heat-inducible DNA binding upon addition of Ref-1 to immunodepleted extracts. The ability of Ref-1 from heated cells to stimulate AP-1 DNA binding was abolished by chemical oxidation and restored by chemical reduction. These results indicate that heat shock activates c-Fos/c-Jun gene expression and AP-1 DNA binding and suggests that redox-sensitive signal transduction pathways involving Ref-1 may mediate heat-induced alterations in AP-1 activation.

Human GABA, Receptor αl and α3 Subunits Genes and Alcoholism

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. GABA effects are largely mediated by binding to the postsynaptic GABAA receptor, causing the opening of an integral chloride-ion channel. The GABAA antagonists picrotoxin and bicuculline reduce some ethanol-induced behaviors, such as motor impairment, sedation, and hypnosis. The role of this receptor in alcoholism is further supported by effective alleviation of alcohol withdrawal symptoms by GABAA agonists. To determine the role of the GABA, receptor (GABR) genes in the development of alcoholism, we have used α1 and α3 simple sequence repeat polymorphisms in a sample of unrelated alcoholics, alcoholic probands with both parents, and psychiatrically normal controls. For the GABRα1 gene, the differences between allele frequencies, when all alleles were compared together, were not significant between total alcoholics, subtypes of alcoholics, and normal controls. However, for GABRα3 , the differences between total alcoholics and normal controls were significant when all alleles were compared together. The differences between subtypes of alcoholics and normal controls were not significant. The results of haplotype relative risk analysis for both genes, GABRα1 and GABRα3 , were also negative. It is possible that the sample size in the haplotype relative risk is too small to have power to detect the differences in transmitted versus nontransmitted alleles. There is a need for a replication study in a large family sample, that will allow haplotype relative risk or affected sib-pair analysis.

Characterization and expression of the mouse Hsc70 gene

A genomic clone encoding the mouse Hsc70 gene has been isolated and characterized by DNA sequence analysis. The gene is approximately 3. 9 kb in length and contains eight introns, the fifth, sixth and eighth of which encode the three U14 snoRNAs. The gene has been located on Chr 9 in the order Fli1-Itm1-Olfr7-Hsc70(Rnu14)-Cbl by genetic analysis. Expression of Hsc70 is universal in all tissues of the mouse, but is slightly elevated in liver, skeletal muscle and kidney tissue, while being depressed in testes. In cultured mouse NIH 3T3 cells or human HeLa cells, Hsc70 mRNA levels are low under normal conditions, but can be induced 8-fold higher in both lines by treatment with the amino acid analog azetidine. A similar induction is seen in cells treated with the proteosome inhibitor MG132 suggesting that elevated Hsc70 expression may be coupled to protein degradation. Surprisingly, expression of the human Hsc70 gene is also regulated by cell-cycle position being 8-10-fold higher in late G1/S-phase cells as opposed to the levels in early G1-phase cells.

The effect of DNA damage on the formation of protein/DNA complexes

Many cellular functions including gene expression and chromosome structure are highly dependent upon the precise recognition and binding of specific DNA elements by regulatory and structural proteins. DNA damage that alters protein/DNA interactions therefore has the potential to disrupt normal cellular functions including proliferation. As a model to examine the interaction of proteins with damaged DNA, the binding of AP-1 transcription factor to cognate DNA elements with 8-oxoadenine, 8-oxoguanine and abasic sites was studied by gel mobility shift analysis. Of the three types of DNA damage only 8-oxoadenine was without effect on AP-1 binding. A single 8-oxoguanine could partially inhibit AP-1 binding when located at specific positions within and even adjacent to the conserved AP-1 binding sequence. Abasic site damage also demonstrated a position effect but with more overall inhibition. When 8-oxoguanine and abasic sites were combined to model the multiple damage sites produced by ionizing radiation there was a cumulative loss of AP-1 binding that appeared to be synergistic. These results suggest protein/DNA interactions can be quite sensitive to the site, degree, and type of DNA damage, even relatively minor modifications.

Redox factor-1 (Ref-1) mediates the activation of AP-1 in HeLa and NIH 3T3 cells in response to heat

The early response genes, c-Fos and c-Jun, are induced by environmental stress and are thought to modulate injury processes via the induction of AP-1-dependent target genes. AP-1 activation is thought to be regulated by changes in intracellular oxidation/reduction reactions involving the redox factor-1 (Ref-1) protein. In this study, NIH 3T3 and HeLa cells were used to determine whether heat shock induces the AP-1 transcription factor via signaling pathways involving Ref-1. Reverse transcriptase-polymerase chain reaction analysis and immunoblotting demonstrated that c-Fos and c-Jun were induced 2-10 h following heat shock, and this induction was accompanied by an increase in AP-1 DNA binding. Electrophoretic mobility shift assay extracts immunodepleted of Ref-1 protein demonstrated that the increase in AP-1 DNA-binding activity following heating was dependent upon the presence of Ref-1 and that Ref-1 regulates inducible, but not basal, AP-1 DNA-binding activity. This was confirmed by the restoration of heat-inducible DNA binding upon addition of Ref-1 to immunodepleted extracts. The ability of Ref-1 from heated cells to stimulate AP-1 DNA binding was abolished by chemical oxidation and restored by chemical reduction. These results indicate that heat shock activates c-Fos/c-Jun gene expression and AP-1 DNA binding and suggests that redox-sensitive signal transduction pathways involving Ref-1 may mediate heat-induced alterations in AP-1 activation.