Calmodulin-like effect of oncomodulin on cell proliferation.

Oncomodulin (OM) is a Ca2+ binding protein (CABP) structurally closely related to parvalbumin. Expression of OM is restricted to early embryonic stages, the placental cytotrophoblasts, and neoplastic tissues. The function of OM as a calmodulin (CaM)-like enzyme modulator is controversial. Two types of experiments demonstrate that OM may act in an analogous fashion to CaM in T14 and T10 cancerous cell lines, which both express OM. First, both OM transcript and protein levels increased at the G1/S boundary in a similar manner to CaM, though not to the same extent, in the chemically transformed rat fibroblast cell line T14 synchronized at mitosis by nocodazole. Second, antisense oligonucleotides specific to the OM ATG region inhibited growth of T14 in a similar dose-dependent manner as observed with CaM-specific antisense probes.

Intracellular Ca2+ and Ca(2+)-binding proteins in chemically transformed rat fibroblasts.

Disturbed Ca2+ handling and altered levels of Ca(2+)-binding proteins (CaBPs) have been found in many transformed cells. To investigate whether there exists a correlation between expression of CaBPs and intracellular Ca2+ we analyzed three transformed rat fibroblast cell lines (T14, T10, and T43) exhibiting different levels of CaBPs. All three cell lines express calmodulin (CaM) at elevated levels compared to normal tissues. In addition, oncomodulin (OM) is expressed at high levels in T14 cells and at much lower levels in T10 cells, while T43 cells do not express OM. We demonstrate that CaBP levels are affected by changes of extracellular [Ca2+] ([Ca2+]o). In reduced [Ca2+]o, CaM levels were increased up to 60%, while OM levels were decreased up to 65% in T14 and T10 cells. Increase of [Ca2+]o, on the other hand, led to a decrease of CaM levels and an increase of OM levels, suggesting that [Ca2+]o exerts its effect on OM and CaM differently. Comparison of the growth rates in reduced [Ca2+]o revealed that T14 cells with the most pronounced CaM increase induced by low [Ca2+]o grew best under these conditions. These results indicate that elevated CaM expression but not the presence of OM reduces the Ca2+ requirement for growth. Intracellular [Ca2+] transients are not buffered by the high concentration of CaBPs in T14 cells. In contrast, [Ca2+]i transients induced by increase of [Ca2+]o or addition of serum were most pronounced in T14 cells compared to T10 and T43 cells.

The induction of DNA strand breaks and formation of semiquinone radicals by metabolites of 2,4,5-trichlorophenol.

The industrial pollutant 2,4,5-trichlorophenol (2,4,5-TCP) was metabolized with postmitochondrial liver fraction from Aroclor-1254 induced rats. The generated metabolites induced single strand breaks in PM2 DNA. Among the metabolites produced are the 3,4,6-trichlorocatechol (TCC) and the 2,5-dichlorohydro-quinone (DCH), whereby the induction of DNA scission by DCH was approximately one hundred times greater than that of TCC. In the 2,4,5-TCP metabolization mixture radicals were observed by ESR. They were identified as the semiquinones of TCC and DCH. ESR studies confirmed that both TCC and DCH autoxidize in aqueous solution to their semiquinone radicals. The involvement of reactive oxygen species in the DNA strand scission was demonstrated by using DMSO, SOD, and catalase as scavengers. Inhibition of strand breaks with the scavenger enzymes did not give homogeneous results for DCH and TCC. This indicated that the directly damaging species might be different for DCH and TCC.

The role of hydroxyl radicals in tetrachlorohydroquinone induced DNA strand break formation in PM2 DNA and human fibroblasts.

Tetrachlorohydroquinone (TCHQ), which has previously been identified as a metabolite of pentachlorophenol, induces DNA strand breaks in isolated DNA and in human fibroblasts. Strand break formation in PM2 DNA is prevented by the addition of catalase and the hydroxyl radical scavengers DMSO, ethanol and mannitol, whereas addition of SOD reduced SSB only slightly. Oxygen radicals are formed by the autoxidation of TCHQ to the tetrachlorosemiquinone radical. Desferrioxamine (0.2 mM) completely abolished strand break formation, whereas the metal chelator DETAPAC (1 mM) reduced SSB by only 8.5%. The formation of the semiquinone radical at physiological conditions is shown by ESR spectroscopy. Exposure of human fibroblasts to TCHQ also leads to DNA single strand breaks measured by the alkaline elution assay. These were reduced by addition of 5% DMSO. This indicates that at least part of the strand break formation in human cells is also due to the action of hydroxyl radicals.

A chemically transformed rat fibroblast cell line expresses high levels of oncomodulin.

Chemically (by N-methyl-N'-nitro-N-nitrosoguanidine) treated rat fibroblasts (T14c) exhibited growth characteristics and a morphology typical for transformed cells and markedly different from untreated, parental cells. In contrast to untransformed cells, T14c fibroblasts produced significant levels of oncomodulin mRNA as analyzed on Northern blots even when compared to rat Morris hepatomas, the richest source of oncomodulin known so far. The levels of transcripts for both calmodulin and oncomodulin in T14c cells were higher in log phase growth as compared to confluent stages. The T14c model system may be useful in the elucidation of mechanisms involved in the regulation of oncomodulin synthesis.