Role of stress responses in human cell survival following exposure to ultraviolet C radiation.

PURPOSE: To investigate in human skin and other cells the role of tyrosine kinase and protein kinase-C (PKC) in eliciting cell-signalling responses to UV radiation (UVR) that affect the survival of irradiated cells. MATERIALS AND METHODS: The survival of HeLa S3 cells, NCTC 2544 human keratinocytes and A431 human epidermal carcinoma cells was measured following incubation with various tyrosine kinase or PKC inhibitors and exposure to UVC (254nm) radiation. In addition, Western blotting measured PKC isozyme expression in human keratinocytes following UVC exposure. RESULTS: It was confirmed that inhibition of tyrosine kinase activation reduces the survival of UV-irradiated HeLa S3 cells. However, no effect was seen on the survival of either NCTC 2544 human keratinocytes or A431 human epidermal carcinoma cells. In contrast, specific inhibition of PKC reduced the survival of UV-irradiated keratinocytes but had no effect on HeLa cells. Comparison of the effects of different inhibitors in keratinocytes suggested that this effect was mediated mostly through PKCmu and PKClambda/iota. In addition, keratinocyte exposure to UVC induced large and temporally distinct increases in PKCmu and PKClambda/iota. CONCLUSIONS: The survival of NCTC 2544 keratinocytes, but not HeLa S3 cells, following UVC exposure is mediated by signalling through PKC, mostly PKCmu and PKClambda/iota. Further study is required to confirm these results in normal human keratinocytes.

50 Hz magnetic field exposure alters onset of S-phase in normal human fibroblasts.

This study was undertaken to investigate whether power frequency magnetic fields can affect the kinetics of cell cycle progression in exposed human cells. To achieve this, cultures of normal human fibroblasts were synchronised in the G(0) phase of the cell cycle and exposed to 50 Hz magnetic fields at a range of flux densities. Progression through the cycle was monitored by examining the timing of entry into S phase, as characterised by the onset of DNA synthesis. Simultaneous positive controls were exposed to human recombinant fibroblast growth factor to demonstrate that the system was responsive to external stimuli. Exposure to magnetic fields at 20 and 200 microT induced a small but significant increase in the length of the G(1) phase of the cell cycle. However, exposure at higher flux densities of 2 and 20 mT had no significant effect. These results are discussed in relation to weak magnetic field effects on free radical concentration.

Effects of 50 Hz magnetic field exposure on the rate of RNA synthesis by normal human fibroblasts.

PURPOSE: To investigate whether exposure to magnetic fields can affect the rate of RNA synthesis, a broad measure of cellular activity. MATERIALS AND METHODS: Normal human fibroblasts were exposed to 50 Hz magnetic fields at a range of flux densities between 2 microT and 20 mT. The rate of synthesis of total RNA was determined by following the incorporation of [3H]uridine into macromolecular material. In addition, polyadenylated RNA was isolated and used to estimate the rate of synthesis of mRNA. RESULTS: Incorporation of [3H]uridine into both total and messenger RNA increased progressively throughout the 5 h exposure period in all cells. However, magnetic field exposure had no detectable effect on the rate of synthesis of either total or messenger RNA when compared with controls. CONCLUSIONS: These findings indicate that under the conditions examined, gross transcription rates are not affected by exposure to power frequency magnetic fields. Taken together with previous data, this suggests that if magnetic fields do alter cellular activity, the effect is likely to be extremely subtle.

Effects of 50 Hz magnetic field exposure on the rate of DNA synthesis by normal human fibroblasts.

Interest in the potential adverse biological effects of exposure to power-frequency magnetic fields has centred on the possibility that these fields may influence tumour promotion, possibly by increasing the rate of cell proliferation. In order to investigate whether exposure to magnetic fields can indeed affect the rate of cell proliferation, normal human fibroblasts were serum starved overnight and then exposed to 50 Hz magnetic fields in a purpose-built facility. The rate of DNA synthesis was taken as a measure of cell proliferation, and was determined by following the incorporation of [3H]-thymidine into macromolecular material. The rate of DNA synthesis in exposed cells was compared with that in control cultures maintained in a standard CO2 incubator where they were exposed to background magnetic fields of < 200 nT. Positive controls were maintained in the same CO2 incubator, but were treated with human recombinant fibroblast growth factor to check that the cells were responsive to growth stimuli. Magnetic fields at 50 Hz and at a range of flux densities between 20 microT and 20 mT had no detectable effect on the rate of DNA synthesis by cells exposed for up to 30 h.

Sunlight-induced mutagenicity of a common sunscreen ingredient.

We have tested the mutagenicity of a UV-B sunscreen ingredient called Padimate-O or octyl dimethyl PABA, which, chemically speaking, is identical to an industrial chemical that generates free radicals when illuminated. It is harmless in the dark but mutagenic in sunlight, attacking DNA directly. A commercial sunscreen containing Padimate-O behaves in the same way. UV-A in sunlight also excites Padimate-O, although less than UV-B. Some related compounds, including a known carcinogen, behave similarly. As mutagens may be carcinogenic, our results suggest that some sunscreens could, while preventing sunburn, contribute to sunlight-related cancers.

Activation of chromosomal vitellogenin genes in Xenopus oocytes by pure estrogen receptor and independent activation of albumin genes.

We generate pure estrogen receptor protein in Xenopus oocytes by injecting them with estrogen receptor mRNA synthesized in vitro. A chromosomal vitellogenin gene, which normally responds to estrogen only in liver cells, is activated. Primer extension shows that initiation is accurate, and ribonuclease mapping shows that the first exon is correctly spliced out of the initial transcript. Long transcripts are produced, one being equal in length to poly(A)- vitellogenin mRNA. Immunochemical estimates of receptor levels in the oocyte nuclei suggest that pure receptor, acting alone, cannot activate oocyte vitellogenin genes unless unusually large amounts are present. However, when a receptor-free extract from liver cells is also injected, the amount of receptor required is reduced. Such an extract, but not pure receptor, can also activate albumin genes in oocytes.

Selective photochemical treatment of oestrogen receptor in a Xenopus liver extract destroys hormone binding and transcriptional activation but not DNA binding.

Photochemical excitation of a simple derivative of oestradiol using light in the UV-A range totally, permanently and selectively inactivates the oestrogen receptor protein present in a Xenopus liver extract without affecting its overall size. Inactivation of the binding site proceeds to completion with simple, first-order kinetics. Inactivation is prevented by excess oestradiol but not by non-oestrogenic steroids. Using an in vitro transcription system, we show that the treatment eliminates transcription of vitellogenin genes, which are normally oestrogen-responsive, but has no effect on the transcription of albumin genes, which are not. Native receptor binds to the two imperfectly palindromic sequences in the vitellogenin B2 gene which together constitute an oestrogen-response unit. Its affinity for one sequence is greater than its affinity for the other, suggesting that a compulsory binding order operates when receptor interacts with the B2 gene. Photoinactivated receptor still binds to both sequences, but with reduced affinity. We also discuss our findings in the context of the current concern over the effects of UV-A on human tissues.