Opposite regulation of the HPV 20-URR and HPV 27-URR promoters by ultraviolet irradiation and cytokines.

Epidemiological evidence implicates ultraviolet radiation and genetic changes (e.g., p53 mutations) as important factors in the etiology of nonmelanoma skin cancer. Little is known about a possible role of cutaneous papillomaviruses in these tumors. We previously reported both positive and negative regulation of the promoter activity of a number of HPV types by UV irradiation. To determine the underlying mechanism, we examined the influence of pro-inflammatory cytokines and MAP-kinases induced by UV irradiation by transfecting the HPV 20-URR and the HPV 27-URR into the RKO, HaCaT and H1299 cell lines expressing wild-type or mutated p53 or lacking p53, respectively. IL-1alpha, IL-1beta, IL-6, IL-17, TNF-alpha, as well as interferon-alpha, -beta and -gamma activated the promoter in the HPV 20-URR but inhibited the HPV 27-URR promoter. The effect of IL-1alpha and UV light was abolished by the addition of IL-1 receptor antagonist. UV irradiation induced a prolonged activation of JNK in HaCaT and H1299 but not in RKO cells, and its dephosphorylation was enhanced in the presence of p53 and the HPV-URRs.

Dipeptidylpeptidase IV activities are elevated in prostate cancers and adjacent benign hyperplastic glands.

Dipeptidylpeptidase IV (DPP IV) is a serine exopeptidase that has been implicated in cell-extracellular matrix interactions and bioactive peptide/cytokine/growth factor metabolism. The objective of this study was to determine if DPP IV activities were changed with development of cancer in the prostate. DPP IV activity was measured in human prostate cancer and benign prostatic hyperplasia (BPH) tissues by biochemical assays with glycylprolyl-p-nitroanalide as substrate in tissue extracts (BPH, n = 8: cancer, n = 7; 2 with Gleason score 5 and 5 with Gleason score 7) and quantitative morphometry of histochemical activities with glycylproline-4-methoxy-beta-naphthylamide as substrate (BPH, n = 9: cancer, n = 13, 1 with Gleason score 4, 10 with Gleason score 6, 2 with Gleason score 8) in frozen-tissue sections. Data were analyzed by analysis of variance. The peptidase activity was detected in epithelial but not stromal cells of BPH and cancer tissues, and it was present as a single band of activity of approximately 160 kDa in electrophoretically separated activity blots of the extracts. DPP IV activity was increased approximately twofold in cancer versus BPH tissues as determined by biochemical and quantitative histochemical methods. In addition, DPP IV activity was increased to a similar extent in BPH glands associated with the cancers. These data indicate that DPP IV activity is increased not only in primary prostatic cancers but also in associated BPH glands, suggesting that there may be some local factors produced by cancer cells that influence adjacent BPH epithelial cells to positively affect the immediate growth environment of the cancer.

Human papillomaviruses in non-melanoma skin cancer.

Recent data suggest that additional factors, other than UV radiation, are involved in the etiology of non-melanoma skin cancer. These include alterations in the tumor suppressor genes, p53, p16$L*I*U$LINK4a$L*I$L/CDKN2A, p21$L*I*U$LWAF1/CIP1$L*I$L and the PTCH gene, as well as cytokines. Papillomavirus infections have been implicated in the etiology of non-melanoma skin cancer. The interaction of tumor suppressor genes and cytokines with the oncoproteins of high-risk mucosal HPV types have been studied in detail, but very little is known about the cutaneous HPV types. We have studied the effect of UV radiation on the URRs of HPV 1, 2, 3, 5, 7, 20, 23, 27, 38, 41, and 77. Neither the CAT-expression and promoter activity of these HPV types, nor presence or absence of wild-type or mutated p53 in the cell lines used, could be related to the DNA sequence homology between the different HPV types or their biological behavior.

Prostate specific origin of dipeptidylpeptidase IV (CD-26) in human seminal plasma.

PURPOSE: A number of peptidases which can metabolize certain bioactive peptides and growth factors have been identified in seminal plasma. Our goal in this study was to determine molecular properties and the tissue source(s) for one of these peptidases, dipeptidylpeptidase IV (DPP IV), in human seminal plasma. MATERIALS AND METHODS: We measured the activities of DPP IV with the dipeptide glycylprolyl-p-nitroanalide and its molecular forms using immunoblotting of seminal plasmas of men who were vasectomized or with different sperm concentrations, and in prostatic and seminal vesicle secretions of men undergoing prostatic surgery. RESULTS: DPP IV in seminal plasma of vasectomized men was a membrane associated dimer comprised of subunits of approximately 110 kDa. Its activity did not differ in seminal plasmas of vasectomized, azoospermic, oligozoospermic and normozoospermic men indicating no correlation with the concentration of sperm originally present in the semen. The DPP IV antigen (CD -26) and enzymic activity were present in prostatic secretion, but absent from that of the seminal vesicles. These data indicate that the prostate gland is the primary source of DPP IV activity in seminal plasma. There was little variation in its activities in repeat seminal plasma samples from the same individual, and there was no change in its activity with age to 50 years. CONCLUSIONS: DPP IV in seminal plasma was derived from the prostate gland and it may be useful as a bioindicator of prostate function and/or disease with age in men.

Genetic characterization of hyperresistance to formaldehyde and 4-nitroquinoline-N-oxide in the yeast Saccharomyces cerevisiae.

The hyperresistance to 4-nitroquinoline-N-oxide (4-NQO) and formaldehyde (FA) of yeast strains transformed with the multi-copy plasmids pAR172 and pAR184, respectively, is due to the two genes, SNQ and SFA, which are present on these plasmids. Restriction analysis revealed the maximal size of SFA as 2.7 kb and of SNQ as 2.2 kb, including transcription control elements. The presence of the smallest 2.7 kb subclone carrying SFA increased hyperresistance to formaldehyde fivefold over that of the original pAR184 isolate. No such increase in hyperresistance to 4-NQO was seen with the smaller subclones of the pAR172 isolate. Disruption of the SFA gene led to a threefold increase in sensitivity to FA as compared with the wild type. Expression of gene SNQ introduced on a multi-copy vector into haploid yeast mutants rad2, rad3, and snm1 did not complement these mutations that block excision repair.

Hyperresistance to DNA damaging agents in yeast.

In order to study resistance to DNA damaging agents, yeast DNA segments conferring hyperresistance in this organism to such genotoxic agents were selected for among yeast cells transformed by a yeast genome library based on the multi-copy vector plasmid YEp13. Genetic variants hyperresistant to 4-nitroquinoline-N-oxide, formaldehyde, and alkylating agents were isolated and the respective hyperresistance determinants shown to co-segregate with the vector plasmid. Phenotypical characterization indicated different degrees of resistance, few cases of cross-resistance and differing structural stability of the cloned DNA. By transfer to E. coli and subsequent retransformation of yeast a number of plasmids was shown to stably carry the genetic information for hyperresistance.

The cytotoxic action of activated and non-activated cyclophosphamide in yeast: comparison of induced DNA damage.

The cytotoxic and DNA-damaging effects of cyclophosphamide (CP) and its 'activated' derivative 4-OOH-CP were studied using a series of strains of S. cerevisiae which allow a phenotypical classification of genotoxic characteristics as well as direct physicochemical demonstration of key DNA lesions. The concurring results of biological and biochemical experiments indicate that (i) non-activated CP has a weak but detectable monofunctional alkylating potency, leading to DNA strand breaks and (ii) 4-OOH-CP has the ability to induce both DNA strand breaks and interstrand cross-links. The activity of CP is probably due to spontaneous decomposition in aqueous solution.

Isolation of yeast mutants sensitive to the bifunctional alkylating agent nitrogen mustard.

Mutants of Saccharomyces cerevisiae with enhanced sensitivity to the DNA cross-linking agent nitrogen mustard (HN2) have been isolated and partially characterized with respect to their phenotypic and genetic properties. The screening technique, based on HN2-sensitivity as sole criterion, yields approxiamtely 1 sensitive isolate in 200 clones when applied to an intensively mutagenized population of a resistant parent strain. Mutants characterized so far are all due to recessive nuclear genes and represent at least seven complementation groups. They exhibit different degrees as well as different patterns of sensitivity towards monofunctional and bifunctional alkylating agents, and ultraviolet light.

Biological and chemical effects of mustard gas in yeast.

Mustard gas induces inactivation and mutation in yeast. Both effects are dose-proportional, indicating single-hit events. Induction of both effects is influenced by the cell's capacity for DNA dark-repair, whereby the probability of reversion is highest in repair-proficient cells. Binding of mustard gas to cells and probably to DNA is independent of DNA-repair systems. The number of inter-strand cross-links, as determined by assaying for renaturability of alkalidenatured DNA, increases in a dose-proportional manner. At 37% survival an excision-deficient strain contains 55 inter-strand cross-links. Chromatographic analysis yields several alkylation products of DNA. Their relative frequencies resemble the values reported for E. coli and bacteriophage T7.

Mutagenesis by cytostatic alkylating agents in yeast strains of differing repair capacities.

Reversion of two nulcear ochre nonsense alleles and cell inactivation induced by mono-, bi-, and tri-functional alkylating agents and by UV has been investigated in stationary-phase haploid cells of yeast strains with differing capacities for DNA repair. The ability to survive alkylation damage is correlated with UV repair capacity, a UV-resistant and UV-mutable strain (RAD REV) being least and a UV-sensitive and UV-nonmutable strain (radi rev3) most sensitive. Mutagenicity of alkylating agents is highest in the former and is abolished in the latter strain. Deficiency in excision repair (rad1 rad2) or in the RAD18 function does not lead to enhanced mutability. Mutagenesis by the various agents is characterized by a common pattern of induction of locus-specific revertants and suppressor mutants. Induction kinetics are mostly linear, but UV-induced reversion in the RAD REV strain follows higher-than-linear (probably "quadratic") kinetics. The alkylating agent cyclophosphamide, usually considered inactive without metabolic conversion, reduces colony-forming ability and induces revertants in a manner similar but not identical to the other chemicals tested. These findings are taken to support the concept of mutagenesis by misrepair after alkylation, which albeit sharing common features with the mechanism of UV-induced reversion, can be distinguished therefrom.

Genetic activity of chemicals in yeast: DNA alterations and mutations induced by alkylating anti-cancer agents.

The simple eukaryotic organism baker's yeast allows demonstration of primary DNA lesions in parallel with measurement of mutagenicity and lethality after treatment with alkylating chemicals. Several anti-cancer drugs formed cross-linked DNA molecules and were genetically active. The mutagenicity and lethality of these drugs varied substantially and were dependent on the function of some processes of DNA dark-repair.