Ultraviolet-B-light-induced mutagenesis of C-H-ras codons 11 and 12 in human skin fibroblasts.

Mutations in the ras oncogene are detected with a high frequency in non-melanoma skin cancer. Approximately half of the squamous-cell carcinomas (SCC) and one third of the basal-cell carcinomas (BCC) carry mutations at the second position of Ha-ras codon 12 (GGC to GTC), whereas mutations in Ki-ras codon 12 occur less frequently. Since the mutations in the Ha-ras and Ki-ras oncogenes are located opposite potential pyrimidine dimer sites (C-C), it is likely that the mutations are induced by ultraviolet radiation present in sunlight. We studied the capacity of ultraviolet B (UVB) light to induce base-pair changes in Ha-ras codons 11 and 12 in human skin fibroblasts. UVB induced mostly C to T and G to A transitions and C to A and G to T transversions. The base-pair change with the highest relative abundance was C to T in the middle position of codon 11 followed by (in diminishing relative abundance) C to A in the middle position of codon 11, G to A and G to T in the middle position of codon 12. The C to T and G to A transitions are compatible with pyrimidine photodimers as pre-mutagenic lesions, whereas the C to A and G to T transversions could be generated due to the formation of 8-hydroxyguanine, which is the major oxidation product of guanine. The relative abundance of mutations induced by UVB in Ha-ras codons 11 and 12 does not correlate with mutations observed in the DNA from non-melanoma skin cancer, where the G to T transversion in the middle position of codon 12 is selected.

Differential cytotoxicity in mouse epidermal JB6 cells: a potential mechanism for oxidant tumor promotion.

It has been suggested that superior antioxidant defense systems protect promotion-sensitive (p+t) mouse epidermal JB6 clone 41 cells from excessive deleterious effects of oxidants, allowing their clonal expansion in contrast to that of promotion-resistant (p-) clone 30 cells. In support of this concept, we report that oxidants produced by xanthine/xanthine oxidase cause more cytotoxicity, cellular damage, and cell death in p-cells. Cell surface blebbing, an early morphological consequence of oxidative injury, was detected in cultures grown on glass coverslips. While a rise in cytosolic ionized calcium ([Ca2+]i) preceding bleb formation was observed in both p+ and p- cells by digital imaging fluorescence microscopy, elevated levels of [Ca2+]i were sustained longer in p- cells. This increase was dependent on the levels of extracellular ionized calcium ([Ca2+]e) in p+ but not p- cells. We conclude that the superior antioxidant defense or improved Ca2+ buffering of promotable clone 41 cells protects them from more severe deregulation of [Ca2+]i and, as a consequence, from excessive cytotoxicity after exposure to oxidant promoters.

Mechanism of induction of c-fos by ultraviolet B (290-320 nm) in mouse JB6 epidermal cells.

The UVB (290-320 nm) portion of the solar spectrum possesses the highest activity for the induction of skin cancer and has the capacity to stimulate epidermal proliferation. We report that UVB is a transcriptional inducer of the c-fos protooncogene in mouse JB6 epidermal cells. Induction is biphasic with an immediate early peak at 30-60 min and a second broader peak 8 h following irradiation. The immediate early phase is suppressed by inhibitors of nuclear adenosine diphosphoribose transferase. For UVB induction, the formation of full-length messages is less efficient than of early, short messages, while both types of messages are produced at similar rates following serum stimulation. Experiments with stable transfectants with reporter constructs linked to 5'-upstream sequences of c-fos indicate that UVB and serum stimulation both require the sequences from -345 to -285 which contain the joint DSE-AP-1 enhancer motifs for efficient induction. Mobility shift data reveal that the complement of c-Fos and c-Jun proteins which bind to the fos-AP-1 octanucleotide decrease immediately following irradiation. Increased binding of Fos and Jun is observed 8-24 h later. UVB did not cause an observable change in the nuclear proteins which bind to the dyad symmetry element oligonucleotide in vitro. Fos protein was detected among the binding proteins. We propose that the two phases of UVB-induced c-fos expression occur by quite different mechanisms. The immediate early phase is inhibited by adenosine diphosphoribose transferase inhibitors because poly-ADP ribosylation of chromosomal proteins is required for the resealing of UVB-induced DNA strand breaks which otherwise retard message elongation. The production of an autocrine factor may be responsible for the late phase of c-fos induction.

Mechanism of c-fos induction by active oxygen.

We have compared the mechanisms of the transcriptional induction of c-fos in mouse epidermal cells JB6 (clone 30) by an extracellular burst of active oxygen of the type produced by inflammatory phagocytes to induction by serum and phorbol ester. All three inducers elicit a characteristic immediate early response of c-fos which is inhibited by the protein kinase inhibitor H7 but enhanced by the protein synthesis inhibitor cycloheximide. Experiments with stable transfectants containing fos 5' upstream regulatory sequences linked to an HSV-tk-chloram-phenicol-acetyl-transferase reporter construct indicate that the joint dyad symmetry element-AP-1 motifs exert the most potent enhancer effect in response to active oxygen as well as serum. It is concluded that the different signal transduction pathways used by these inducers converge to the same 5' regulatory sequences of c-fos. In contrast to these common features only active oxygen induction of c-fos required the poly-ADP-ribosylation of chromosomal proteins. The inhibitors of ADP-ribose transferase benzamide and 3-amino-benzamide suppressed the elongation of the c-fos message and the de novo synthesis of nuclear factors, among them c-Fos and c-Jun, which bind to the fos-AP-1 motif in vitro only following stimulation with active oxygen. No active oxygen-induced change was observed in the protein complex which binds to an oligonucleotide containing the SIF and dyad symmetry element motifs in vitro. The presence of Fos and Jun proteins was detected in this complex. Only active oxygen, but not serum or phorbol ester, induces DNA breakage. We propose that poly-ADP-ribosylation is required because it participates in the repair of DNA breaks which interfere with transcription. We observed that Fos protein is weakly poly-ADP-ribosylated in response to active oxygen, but the functional role of this modification remains unclear.

Genotypic analysis of N-ethyl-N-nitrosourea-induced mutations by Taq I restriction fragment length polymorphism/polymerase chain reaction in the c-H-ras1 gene.

In genotypic mutation analysis DNA sequence changes are determined without the in vivo or in vitro selection of phenotypically altered cells. We have studied the induction of base-pair changes by N-ethyl-N-nitrosourea in Taq I endonuclease recognition site 2508-2511 (TCGA) of the c-H-ras1 gene in human fibroblasts by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) method. This site contains the four bases, and all 12 possible single base-pair changes can be monitored. The transition of guanine to adenine at position 2510 was the major mutation detected by lambda plaque oligonucleotide hybridization and quantitative sequence analysis of the RFLP/PCR products. It involves the G residue of the CpG sequence of the coding strand. Data calibration with an internal mutant standard indicates that absolute frequencies for this transition lie in the range of 4-12 x 10(-7). The present study documents the capacity of the RFLP/PCR approach to measure mutagen-induced base-pair changes in a specific gene sequence without the selection of a phenotypically altered cell.

Genotypic analysis of mutations in Taq I restriction recognition sites by restriction fragment length polymorphism/polymerase chain reaction.

Point mutations in somatic cells play a role in the etiology of several classes of human pathologies. Experimental procedures are required that allow the detection and quantitation of such mutations in disease-related genes in tissue biopsy samples without the need for the selection of mutated cells. We describe the genotypic analysis of single base pair mutations in the Taq I endonuclease recognition sequence TCGA, residues 2508-2511 of exon 2 of the human c-H-ras1 gene, by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) approach. The high thermostability of Taq I endonuclease allows the continuous removal of eventual residual wild-type sequences during the thermocycling of the PCR and reduces polymerase errors in the final RFLP/PCR product to a minimum. As few as five copies of a mutant standard containing two base pair changes in the chosen Taq I site could be rescued from 10(8) copies of wild-type DNA. Taq I RFLP/PCR holds promise for the monitoring of mutations in biochemical epidemiology.

Inflammation and oxidative stress in carcinogenesis.

Oxidants, which are generated by multiple pathways in mammalian organisms, may be natural carcinogens and contribute to several stages of malignant transformation. Active oxygen released by inflammatory phagocytes and more stable "clastogenic factors" can induce mutations and chromosomal aberrations in neighboring target cells. These oxidant-induced DNA sequence changes, though rare, may affect the activities of proto-oncogenes and suppressor genes. In addition, oxidants can promote cell growth. Like polypeptide growth factors they activate kinases. Because they break DNA, they also induce the poly ADP-ribosylation of chromosomal proteins. Both phosphorylation and poly ADP-ribosylation appear to participate in the transcriptional induction of the growth-related proto-oncogene c-fos. Growth stimulation by oxidants is modulated by the cellular antioxidant defenses. Maximal growth promotion is observed when cells are protected from excessive toxicity but still maintain a sufficient oxidant signal for the induction of growth-competence genes.

c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy.

Spontaneously immortalized human skin keratinocytes (HaCaT) were transfected with the c-Ha-ras (EJ) oncogene via a plasmid construct which also contained the selectable neomycin gene. Clones were selected on the basis of G418 resistance. Those clones that had stable integrants of Ha-ras fell into 3 classes with respect to tumorigenicity. Class I clones were nontumorigenic, i.e., formed nodules which rapidly regressed. This phenotype is identical to that seen with parental HaCaT cells. Class II clones formed slowly growing, highly differentiated cystic or papillomatous-type benign tumors, and class III clones formed highly differentiated, locally invasive squamous cell carcinomas. The clones of all three classes exhibited similar morphology and growth potential in culture and retained the ability to reconstitute an epidermis-like stratified epithelium in transplantation experiments. Only the malignant clones showed locally invasive growth. Both the benign and the malignant clones exhibited higher levels of ras integration and variable levels of mutated p21 protein product. Thus, expression of the cellular Ha-ras oncogene in these human epithelial cells significantly altered growth regulation, resulting in varying degrees of growth potential in vivo, ranging from benign to malignant tumors. However, no direct correlation was seen between high levels of p21 expression and malignant growth.

UV-enhanced reactivation of UV-damaged SV40 is due to the restoration of viral early gene function.

Mammalian cells respond to UV-radiation by inducing an increased ability to support the survival of UV-damaged virus. We have tested whether the induction of enhanced viral reactivation (ER) reflects heightened UV-resistance of specific viral functions. For this, we examined the extent of ER for SV40 containing UV-damage in three functionally distinct regions of the SV40 genome: (i) the viral regulatory region, (ii) the early genes region and (iii) the late genes region. ER corresponding to a dose reduction factor of 43% was observed for damage in the early genes region. No ER was observed for damage in the regulatory or late genes regions. We conclude that ER in SV40 reverses the lethal disruption of an essential function peculiar to the viral early genes region. This function is almost certainly transcription.

Response modification in carcinogenesis.

A major goal in multistep carcinogenesis research is the integration of recent findings obtained by sophisticated molecular-genetic and cytogenetic analysis of cancer into the more descriptive concepts of experimental pathology. It is proposed that the creation of a promotable cell in carcinogenic initiation requires a response modification to extracellular or intercellular signals. Different types of response modification can be distinguished: changes in the receptors for growth and differentiation factors and their cytoplasmic and nuclear signal transduction pathways; increased resistance of initiated cells to cytotoxic agents; alterations in junctional cell-to-cell communications. The challenge of a response-modified cell to an appropriate promoter results in its selection and clonal expansion, usually to a benign tumor. In addition, for malignancy, chromosomal changes are required that affect cellular functions that can play a role early or late in tumorigenesis. These concepts are illustrated with examples from oncogene research and oxidant promotion.

Modified SV40 for analysis of mismatch repair in simian and human cells.

We have developed a way of introducing specific mispairs into the genome of simian virus 40 and of determining the fate of the mispaired bases in simian and human cells. Mispairs are introduced into viral DNA within the intron of the gene coding for the large T antigen. Each DNA molecule harbors a single mispair in a defined orientation. Transfection of mismatch-containing SV40 DNA into host cells yields plaques, each corresponding to a productive infection initiated by a single viral DNA molecule. Isolation of DNA derived from individual plaques and determination of the DNA sequence at the site of the mispair reveals whether correction occurred and what the repair products are. Here we describe repair patterns for G/T and A/C mispairs in CV-1 African green monkey kidney cells, and for G/T mispairs in human fibroblasts derived from 3 normal individuals, 1 patient with xeroderma pigmentosum (complementation group A), and 3 patients with Bloom's syndrome. G/T mispairs, which arise in resting DNA through the deamination of 5-methylcytosine (mC) to form thymine, are corrected in all cases with extremely high efficiency and nearly always in favor of guanine. In contrast, A/C mispairs are corrected randomly and relatively inefficiently in simian cells.

Mechanisms of action of oxidant carcinogens.

Oxidants can act at multiple stages of carcinogenesis. While they cause genetic damage and are cytotoxic, they also activate cellular pathways which alter gene expression, growth, and differentiation. Certain pathways used by polypeptide growth factors and hormones are also activated by oxidants. For example, oxidants stimulate the phosphorylation of the ribosomal subunit S6, the phosphotransferase activity of protein kinase C, and induce its translocation to the plasma membrane. On the genomic level, oxidants increase the transcription of the growth-competence-related protooncogenes c-fos and c-myc. In addition to these growth factor-type reactions, oxidants induce pathways which are unique to them. Poly ADP-ribosylation of chromosomal proteins is of particular relevance to oxidant carcinogenesis. It represents an epigenetic consequence of DNA-breakage. Both histones and nonhistone proteins are poly ADP-ribosylated in response to oxidants. Among non-histones, ADPR-transferase, topoisomerase I, and the fos oncoprotein were identified as acceptors. Inhibition of poly ADP-ribosylation suppressed the oxidant-induced transcription of c-fos. Since fos oncoprotein serves as a transcriptional regulator, we speculate that its poly ADP-ribosylation and that of other chromosomal proteins plays a role in the modulation of gene expression in response to oxidative stress.

Differential effects of glutathione depletion and metallothionein induction on the induction of DNA single-strand breaks and cytotoxicity by tert-butyl hydroperoxide in cultured mammalian cells.

Induction of DNA single-strand breaks (ssb), their resealing and cytotoxicity by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. The effect of the depletion of cellular glutathione (GSH), iron chelation and induction of metallothionein (MT) on these parameters was studied. t-BuOOH in a concentration range of 0.02-0.5 mM induced DNA ssb in a dose-dependent fashion. Strand breakage increased as a function of time up to 1 h. Divalent iron chelator o-phenanthroline suppressed markedly the induction of DNA ssb while the trivalent iron chelator desferrioxamine had no effect. GSH-depletion increased cytotoxicity of t-BuOOH. In contrast, the depletion of GSH did not affect the efficiency of formation of DNA ssb by t-BuOOH and the rate of resealing of the DNA damage. The induction of MT did not influence the efficiency of formation of DNA ssb by t-BuOOH. In summary, while GSH depletion and MT induction affected the formation of DNA ssb and cytotoxicity differently divalent iron was required for both. Therefore, appears likely that DNA breakage and cytotoxicity by t-BuOOH are caused by independent mechanisms.

Constitutive and phorbol-myristate-acetate regulated antioxidant defense of mouse epidermal JB6 cells.

Because oxidative processes can participate in tumor promotion, it is likely that the cellular antioxidant defense also plays a role. We have compared the levels of the three major antioxidant enzymes, Cu,Zn-superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx), in promotable mouse epidermal JB6 cells clone 41 and nonpromotable cells, clone 30. We found that the constitutive activities of SOD and catalase were approximately twice as high in clone 41 as in clone 30 while the GPx activities were comparable. Correspondingly, catalase protein concentrations were higher in clone 41, according to immunoblots. Northern blot analysis indicated that the steady-state mRNA concentrations for SOD and catalase, but not for GPx, were considerably higher in clone 41 than in clone 30. Southern blot analysis showed no difference between the two clones in their complements of the SOD and catalase genes. Clone 41 also contained slightly higher constitutive levels of glutathione. The higher antioxidant capacity of promotable clone 41 may protect it from excessive toxicity of oxidant promoters and allow growth stimulation. Certain tumor promoters that lack oxidizing properties may generate a cellular prooxidant state by a variety of mechanisms (e.g., it had been reported that the phorbol ester PMA decreases the activities of catalase and SOD in mouse skin). We found for JB6 cells that this loss of enzyme activity was due to a decrease in the steady-state concentrations of catalase and SOD mRNA. No significant changes in the rates of transcription were detected in nuclear run-off experiments. The observed decreases in catalase and SOD can be considered as part of the complex reprogramming of gene expression that is set in motion by phorbol-12-myristate-13-acetate.

A comparison of markers of human fibroblast transformation induced by chemical carcinogen treatment or by transfection of an origin-defective SV40-containing plasmid.

We have investigated the sensitivity to oncogenic transformation by an origin-defective SV40-containing plasmid, '8-16' (ori-SV40), of skin fibroblasts from normal individuals (NF), and from patients with 2 hereditary diseases characterized by an increased cancer risk, Bloom's syndrome (BS) and Fanconi's anemia (FA). It was hypothesized that perhaps these cells had already undergone some stage, or stages, of the progression to neoplasia, and that as a consequence of these changes, one could observe differential expression of characteristics of the transformed phenotype in these cells compared to normal, or perhaps they would behave differently in vivo. The data showed that FA cells and NF possessed comparable sensitivities to transformation by ori-SV40 DNA transfection, as measured either by focus formation above a confluent monolayer, or anchorage-independent growth. The BS cells, on the other hand, were 5-10 times less sensitive to this method of transformation, and further, the transformed phenotype was unstable. The resistance of BS cells to transformation by the 8-16 plasmid may be a reflection of their inherent genetic instability which affects stable integration and expression of the transfected plasmid DNA, since no differences in initial uptake of transfected DNA were observed between the various cell strains. Immortality and tumorigenicity were not readily demonstrated in this ori-SV40 transformation model. The results are discussed in relationship to the characteristics of the transformed phenotype of chemically treated normal human fibroblasts. SV40, an agent known to transform human cells, can be cast in a positive control role with respect to the appropriateness of the assays, the frequency of appearance of various markers, immortality and tumorigenicity. The tumorigenicity results are further compared to results obtained during the establishment of a wide range of fresh human tumor biopsies as xenograft lines in athymic nude mice, with particular emphasis on the sarcoma data.

Molecular defect in human acatalasia fibroblasts.

The human hereditary disease Acatalasia (AC) is characterized by low or no catalase activity in all body tissues. We have studied the molecular basis of AC. In order to assess their antioxidant defense status we measured the enzyme activities, protein levels and m-RNA concentrations of catalase, superoxide dismutase and glutathione peroxidase in fibroblasts from a Japanese (AC65) and a Swiss (AC64) patient and several normal individuals. Our results point to genetic heterogeneity. While strain AC64 contained normal levels of catalase mRNA and -protein, strain AC65 was completely devoid of both. A structural mutation in the catalase gene is probably responsible for the inactivation of the enzyme in AC64. Since AC65 contains at least a major portion of the catalase gene it may represent a regulatory mutation in which the gene is not transcribed.

Molecular mechanisms involved in T cell activation. III. The role of extracellular calcium in antigen-induced lymphokine production and interleukin 2-induced proliferation of cloned cytotoxic T lymphocytes.

The role that extracellular calcium plays in activating resting cloned cytotoxic T lymphocytes (CTL) to proliferate and to produce lymphokines was examined. In these cells, stimulation with interleukin 2 (IL-2) induced a proliferative response without a concomitant production of macrophage-activating factor (MAF), whereas stimulation with antigen or lectin (in the absence of IL-2) induced MAF production but not proliferation. In the case of IL-2-induced proliferation, extracellular calcium was required to initiate proliferation as well as to prevent cellular arrest later in the G2 + M phase of the cell cycle. In MAF production extracellular calcium was required both to activate the phosphatidylinositol signal-transducing mechanism and to mobilize intracellular calcium in antigen- or lectin-stimulated cytotoxic T lymphocytes. Further, extracellular calcium was required for only 8 of the 18 hr of stimulation time which was needed to achieve maximal MAF production, indicating that both calcium-dependent and -independent events exist in the signal pathway. Additional experiments with calcium ionophores and activators of protein kinase C indicated that although both intracellular calcium mobilization and de novo protein phosphorylation are involved in MAF production, an optimal increase in the level of intracellular calcium by itself is insufficient to induce the production of this lymphokine.