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A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p60gag-cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3' end in the last coding exon, which probably resulted in a deletion and an altered C' terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor.

Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.

Inhibition by alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638) of tyrosine-specific protein kinase was examined using epidermal growth factor (EGF)-treated A431 cells at the concentration of 25 to 100 microM. ST 638 had negligible effects on the growth and morphology of A431 cells and on EGF binding to its receptor, and subsequent down-regulation of the receptor. ST 638 specifically inhibited EGF-induced phosphorylation of tyrosine residues of whole cell proteins in a dose-dependent manner without affecting the phosphorylation of serine and threonine residues. ST 638 greatly inhibited the EGF-induced phosphorylation of lipocortin I at 25 microM, and yet had a negligible effect on the EGF-induced phosphorylation of EGF receptor. Neither the amount of [35S]methionine-labeled lipocortin I nor the serine/threonine phosphorylation level of fodrin beta-subunit was affected by the same concentration of ST 638. These results indicate that the phosphorylation of lipocortin I is not relevant to the transformation of A431 cells. In cell lines transformed by src or fgr oncogene encoding tyrosine kinase, ST 638 also inhibited phosphorylation of calpactin I (p36) without affecting that of the oncogene products. Two-dimensional polyacrylamide gel electrophoresis showed that ST 638 specifically inhibited the EGF-induced phosphorylation and dephosphorylation of cellular proteins in A431 cells.

Two non-tumorigenic variant cells were isolated from UV-irradiated Balb/c 3T3 cells on the basis of their different responsiveness in phorbol ester-induced morphological change (rounding formation). They showed marked differences of lung metastatic potentials after intravenous injections of their v-src transformants into nude mice; phorbol ester-resistant variant TR4 cells transformed by v-src were hypermetastatic, whereas v-src transformants of phorbol ester-sensitive variant TR5 cells were not metastatic at all. These different metastatic responses were not observed in v-K-ras-induced transformants of the variants. These non-tumorigenic variant cells may pre-acquire the genetic alteration of certain src-specific and metastasis-associated factors. This system may be useful for genetic analysis of the induction of metastasis.

The Syrian hamster embryo cell lines, SHOK and MC-1, were used as recipient cells for DNA transfection assay to detect transforming genes in experimental mouse tumours. A mouse repeat sequence was utilised to check whether each transformed focus included mouse genomic DNA in the Hamster background. We investigated five mouse tumours that are related to X-ray radiation, and detected activated c-K-ras, c-mos, and c-cot oncogenes which induced foci of hamster cells. These results show that SHOK and MC-1 cells have unique properties for detecting transforming genes in experimental mouse tumours. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.

We have determined the nucleotide (nt) sequence of 5.5 kb including the 5' flanking, first untranslated exon and first intron regions of the human smooth muscle (SM) (aortic type) alpha-actin-(Sm alpha A)- encoding gene. The promoter region and a part of the first intron show remarkably high sequence conservation with equivalent regions of the chicken gene, and contain multiple transcriptional regulatory elements. From transient chloramphenicol acetyltransferase gene (cat) expression assays in SM cells, a DNA fragment from nt -123 to +49 containing two CArG boxes showed strong positive promoter activity, whereas a far upstream region from nt -253 to -124 showed a negative effect. The conserved region in the first intron also contains the CArG box and showed an enhancer activity. Therefore, the human SM alpha A gene is controlled under positive and negative mechanisms.

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.

We established a subclone, SHOK, from the GHE-L cell line, an immortal line derived from a primary culture of Syrian hamster embryo cells, as a recipient cell line useful for the detection of oncogenes by transfection. SHOK cells were almost as susceptible as NIH 3T3 cells to focus formation by many oncogenes, including v-raf, v-Ha-ras, v-Ki-ras, or activated c-Ha-ras. The susceptibility of SHOK to focus formation was higher than that of NIH 3T3 for v-mos but was lower for v-fps, v-fgr, v-src, v-sis, and v-abl. When DNAs extracted from 27 human and murine tumors were tested for focus formation, 5 DNAs were positive in NIH 3T3 cells, whereas 9 were positive in SHOK cells at the primary transfection. Using SHOK cells as recipients of tumor cellular DNA, we isolated another oncogene and a c-Ki-ras2 gene mutated at codon 146 that were difficult to detect in NIH 3T3 cells. SHOK cells have a low rate of spontaneous transformation, produce easily distinguishable foci, and maintain a stable karyotype in transformed cells. In addition to being useful for the screening of human tumor DNAs, SHOK cells will be useful for the isolation of oncogenes from murine tumors because of their hamster origin.

We investigated N-ras activation in childhood acute lymphoblastic leukemia (dALL) by the polymerase chain reaction (PCR) and the oligonucleotide hybridization method. The frequency of point-mutation of the N-ras gene was not high (2 of 15), and one positive case who relapsed was analyzed in detail. Although N-ras gene activation was detected at both onset and relapse, the mutation sites were different. At onset, Gly (GGT) was changed to Ser (AGT) at codon 12, and at relapse, Gly (GGT) to Asp (GAT) was observed at the same codon. In addition, the DNA at relapse showed a remarkably higher transforming activity than the DNA at onset on two independent recipient cell lines. The identical cell surface phenotype and the same rearrangement patterns of both the immunoglobulin (Ig) heavy chain and T-cell receptor (TCR) gamma chain genes indicated that the leukemic cells at onset and those at relapse were derived from the same precursor cell. Therefore, this case supports the concept that ras activation is not the event initiating leukemogenesis, but may be involved in leukemic progression.

Transcription of the {beta}-actin gene is rapidly inducible in response to serum stimulation. To determine the regions responsible for serum inducible and basal level expression, the human {beta}-actin promoter was subjected to mutational analysis. Two distinct elements, the CCAAT homology and the {beta}-actin specific conserved sequences, were found by a chloramphenicol acetyltransferase expression assay and sequence comparisons, and then analyzed for possible functions. Using a DNA bend assay, it was shown that the conserved sequences included the core of a sequence-directed bend of DNA. Gel mobility shift and DNase I protection assays revealed that the conserved sequences and the CCAAT homology were recognized by binding factors in HeLa cell extracts.

Cultured fibroblast cells derived from a skin biopsy sample taken from normal human adult were exposed to a potent carcinogen, 4-nitroquinoline 1-oxide. Alterations of cell growth pattern such as higher density and piling up of cells were noticed in some fractions of cultures that were successively subcultured after nitroquinoline oxide treatment. Morphologically altered cells retained this growth pattern and became established lines of transformed cells without showing the limited life-span characteristic of normal cells in culture. The transformed cells showed a higher saturation density and the ability to grow in soft agar, properties that are usually correlated with neoplastic transformation of cells in culture. Selection of preexisting transformed human cells as a mechanism of this observed transformation seemed unlikely because clones of these normal cells could also be used to assess the transforming effect of nitroquinoline oxide. Preliminary results suggest that numerous cell divisions were required for the development of the transformation after nitroquinoline oxide treatment of these human cells. When the transformed cell lines were injected subcutaneously into nude (athymic) mice, solid tumors were produced at the site of inoculation. Treatment with N -methyl- N ′-nitro- N -nitrosoguanidine also induced cell transformation, in a manner similar to treatment with nitroquinoline oxide. However, transformation was not induced with ( i ) 4-aminoquinoline 1-oxide (a noncarcinogenic derivative of 4-nitroquinoline 1-oxide), ( ii ) 3-methylcholanthrene (a carcinogen that cannot be metabolically activated by the target cells employed), or ( iii ) the solvent dimethyl sulfoxide.

By in vitro translation, we have identified the mRNA species that codes for a novel actin polypeptide (Ax-actin) in the chemically transformed human fibroblast line HuT-14. The relatedness of the coding sequences of the Ax- and beta-actin genes is indicated by our finding that pcDd actin ITL-I DNA, a recombinant plasmid DNA that contains a DNA sequence complementary to actin mRNA of Dictyostelium discoideum, hybridizes both the Ax-actin mRNA and the beta-actin mRNA but not the gamma-actin mRNA. In contrast, pcHa-1 DNA, a recombinant plasmid constructed by cloning a DNA sequence complementary to human actin mRNA from HuT-14 cells into pBR322, hybridized to all three mRNA species. In addition, no difference was observed between Ax- and beta-actin mRNAs when their molecular size was determined either by sucrose density gradient sedimentation or by methyl mercury agarose gel electrophoresis. Southern blot transfer of radioactive pcDd actin DNA to restriction endonuclease-digested Hut-14 DNA produced only a single hybrid band (a 6-kilobase fragment); the pcHa-1 DNA probe detected one additional band (a 3-kilobase fragment). These results suggest that HuT-14 cells contain only one copy per haploid genome for Ax- or beta-actin. When considered together with recent determination of the entire amino acid sequences of Ax- and beta-actin, our findings indicate that Ax-actin is the product of a mutated beta-actin gene and are evidence for the occurrence of a mutation in a chemically transformed cell.

By Southern blotting and hybridization analysis using 32P-labeled poly(dT-dG) . poly(dC-dA) as a probe, we have found, in eukaryotic genomes, a huge number of stretches of dT-dG alternating sequence, a sequence that has been shown to adopt the Z-DNA conformation under some conditions. This sequence was found in all eukaryotic genomes examined from yeast to human, indicating extraordinary evolutionary conservation. The number of the sequence ranged from about 100 in yeast to tens of thousands in higher eukaryotes. Comparison of nucleotide sequences of dT-dG alternating regions and its flanking regions in several cloned genes showed that the repeated element [the Z(T-G) element]] consists only of dT-dG alternating sequence with variable length. The presence of another purine-pyrimidine alternating sequence was also surveyed in eukaryotic genomes by Southern blot hybridization using 32P-labeled poly(dG-dC) . poly(dG-dC) as the probe. The stretches of dC-dG alternating sequence [the Z(C-G) element] were found to be moderately repetitive in human, mouse, and salmon genomes. However, a few and no copies of the Z(C-G) element were found in yeast and calf genomes, respectively. These results provide evidence for the abundance of potential Z-DNA-forming sequences in nature.

The expression of actin was examined and compared in several mouse B16 melanoma cell lines with different metastatic ability, by the use of two-dimensional gel electrophoresis or horizontal isoelectric focusing. In the mouse B16 melanoma cell lines, the expression of newly found AX actin (Mr = 43,000, pI = 5.2) decreased with the increase in in vitro and in vivo selection cycles (F number) for high-metastatic cells. On the contrary, the metastatic ability of each mouse cell line, assessed by lung colony-forming ability following iv administration, increased with increase in the F number. The half life of AX actin was much the same as that of beta- and gamma-actin and the different expressions of AX actin between the low- (F = 1) and high-metastatic (F = 10) cell lines were attributed to differences in the rate of synthesis but not in the decay rate of AX actin. The AX actin was incorporated into the cytoskeletal fraction with the same efficiency as beta- and gamma-actin. The invasiveness of the cells, assessed in vitro using matrigel, was increased with the decrease in AX expression. The actin stress fibers, observed staining with rhodamine-conjugated phalloidin, were organized better in a low-metastatic cell line (F = 1) than in a high-metastatic one (F = 10). These results suggest to us that depression of AX actin is involved in disorganization of the cytoskeletal system, the cellular flexibility and motility are enhanced and there is a consequent increase in the invasiveness and metastatic potential.

The motility of individual mammalian cells is crucial for many biological processes. This report describes a new technique to quantitate cell motility, momentary alterations of cell shape, based on trace images obtained by video-image analyses and computer techniques. By means of this system, quantitation of cell motility could be automatically done without human observation or subjective judgement. Quantitative data from transformed and nontransformed rodent fibroblasts revealed that the cell motility measured here was related to the expression of such transformed phenotypes as morphological changes and tumorigenicity.

Two distinct 68-kDa proteins, named 68K-I (pI 6.4) and 68K-II (pI 5.6), were solubilized from human placenta by treatment with 5 mM EGTA. On DE52 cellulose column chromatography at pH 7.4, 68K-I in the EGTA eluate was recovered in the unadsorbed fractions, whereas 68K-II was retained on the column and eluted with 0.2 M NaCl. The 68K-I protein was obtained in more than 95% purity by further hydroxylapatite and cation exchange chromatographies, while the 68K-II protein was purified further by gel filtration and hydroxylapatite chromatographies. Partial amino acid sequence data showed that 68K-I protein was a novel protein which shared the same sequences as lipocortin I and that 68K-II was the same as human p68/67-kDa calelectrin (Crompton, M. R., Owens, R. J., Totty, N. F., Moss, S. E., Waterfield, M.D., and Crumpton, M. J. (1988) EMBO J. 7, 21-27; Südhof, T. C., Slaughter, C. A., Leznicki, I., Barjon, P., and Reynolds, G. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 664-668). The two proteins bound to acidic phospholipids, phosphatidylserine, and/or phosphatidylinositol, but not to phosphatidylcholine, in the presence of micromolar levels of Ca2+. 68K-I bound to phosphatidylinositol preferentially to phosphatidylserine, whereas 68K-II bound only to phosphatidylserine. Both 68K-I and 68K-II inhibited phospholipase A2 activity, and the inhibition by 68K-II was detectable only in the presence of 100 mM KCl. 68K-I, but not 68K-II, was found to bind to F-actin in a Ca2+-dependent (1 mM) manner. Moreover 68K-I, but not 68K-II, was phosphorylated in vitro at tyrosine residues by fps kinase and by epidermal growth factor receptor/kinase, the latter reaction being dependent on Ca2+ and epidermal growth factor. Western blot analysis with affinity purified anti-68K-I and anti-68K-II antibodies showed that 68K-I was located in only certain tissues, especially human placenta, whereas 68K-II was present in many human and rat tissues.

Abnormal expression of various oncogenes has been implicated in the development of many malignant tumors. Although RNA blotting methods have been used to measure abnormal expression, they involve the time-consuming process of individually labeling the oncogene probes. To simplify this process we have attempted to develop a new method, termed simultaneous screening, which is based on the synthesis of radiolabeled cDNA corresponding to the mRNA population of malignant cells and on hybridization with various oncogene probes, immobilized on a membrane filter. This method circumvents the time-consuming process of the prevailing RNA blotting methods and is also sensitive enough to detect accurately a five- to ten-fold level of expression of rare mRNA (approximately 10 copies per cell). Overexpression of ten oncogenes was detected in a variety of malignant cells and mitogen-stimulated cells with this method. These results suggest that our simultaneous screening method can be used to examine the overexpression of oncogenes.

An enhancer of the human beta-actin gene and a factor that specifically interacts with it were detected. A mobility shift assay showed that the factor bound to the 25-base-pair sequence (between +759 and +783 downstream from the cap site) with high specificity. This finding correlated with those of DNase I protection and exonuclease III digestion assays. This binding region of the beta-actin enhancer contained a hyphenated dyad symmetry and an enhancer core-like sequence. In vitro competition experiments indicated that the factor did not bind to the simian virus 40 enhancer core region.

Two recombinant phages that contain cardiac muscle actin gene were isolated from a human DNA library and their structures were determined. Restriction analysis indicates that both clones carry the same EcoRI 13-kilobase fragment where the coding sequence is mapped. The cloned DNA hybridized with polyadenylylated RNA from human fibroblasts, which directs the synthesis of cytoplasmic beta- and gamma-actin in vitro. However, sequence determination of the cloned DNA showed that the entire coding sequence perfectly matched the amino acid sequence of cardiac muscle actin. The initiation codon is followed by a cysteine codon that is not found at the amino-terminal site of any actin isoform, suggesting the necessity of post-translational processing for in vivo actin synthesis. There are five introns interrupting exons at codons 41/42, 150, 204, 267, and 327/328. Surprisingly, these intron locations are exactly the same as those of the rat skeletal muscle actin gene but different from those of nonmuscle beta-actin gene. Nucleotide sequences of all exon/intron boundaries agree with the G-T/A-G rule (G-T at the 5' and A-G at the 3' termini of each intron). The 3'-untranslated sequence has no homology to that of nonmuscle beta- or gamma-actin gene, but Southern blot hybridization has shown that this region has considerable homology to that of one of the other actin genes. These results indicate that the recombinant phages, which we have isolated, contain cardiac muscle actin gene and that cardiac muscle actin gene and skeletal muscle actin genes are derived from their ancestor gene at a relatively recent time in evolutionary development.

A recombinant phage that carries the cytoplasmic beta-actin gene was isolated from a human DNA library. The nucleotide sequence of this gene was determined. The amino acid sequence deduced from the nucleotide sequence matches perfectly that of beta-actin from human fibroblasts. The gene contains five introns. A large intron was found in the 5' untranslated region six nucleotides upstream from the ATG initiation codon. Four introns were found within the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. In contrast to the human cardiac muscle actin gene, the aorta-type smooth muscle actin gene, and the stomach-type smooth muscle actin gene, the beta-actin gene lacks the codon for cysteine between the ATG initiation codon and the codon for the NH2-terminal amino acid of the mature protein. Hybridization of genomic DNA with DNA fragments derived from intron I in the 5' untranslated region and from intron III strongly suggests the presence of a single beta-actin gene in the human genome. The DNA sequences of the coding region, of the 3' untranslated region, and of the sequence block between the "CCAAT" box and "TATA" box in the 5' flanking DNA of the human beta-actin gene are highly homologous to the corresponding sequences of the rat and chicken beta-actin genes. Unexpectedly, the sequence of intron III of the human beta-actin gene shows considerable homology to that of the rat beta-actin gene.

Addiction to morphine is a problem widely studied but not yet solved. One of the difficulties of this problem is that it involves physical and psychic factors and it is believed that these signs can be demonstrated only in vertabrates with highly developed cerebral hemispheres. Consequently, addiction experiments in animals have been carried out with higher animals, such as man, monkey, chimpanzee and dog. However, it has been recently reported that rats dosed morphine at 12 hours intervals for several weeks show signs of physical dependence to morphine (1-5). However, we have as yet very little information as to the possibility that similar signs can be also demonstrated in mice. A study of addiction or physical dependence in mice as compared with rats, namely the species difference between mice and rats in their response to morphine, is of great importance for the elucidation of the mechanism of the action of analgesics. The present paper deals with the development of tolerance to and the occurrence of physical dependence to morphine in mice.

Teleocidin, which was isolated from mycelia of Streptomyces, is a potent tumor promoter in mouse skin. The catalytically hydrogenated compound dihydroteleocidin B markedly enhanced malignant cell transformation induced by 3-methylcholanthrene or ultraviolet radiation. Dihydroteleocidin B was at least 100 times more effective in enhancing transformation than 12-O-tetradecanoyl phorbol-13-acetate, the strongest promoter known until now, whereas both promoters showed equal capacities to induce early membrane effects and DNA synthesis.

Aplysiatoxin and debromoaplysiatoxin, which are isolated from the seaweed, Lyngbya gracilis, differ in their chemical structure only by the presence or absence of a bromine residue in the hydrophilic region. The function and the structure-activity relation of the hydrophilic region are not known. Aplysiatoxin increased malignant transformation, stimulated DNA synthesis, and inhibited the binding of phorbol-12,13-dibutyrate and epidermal growth factor to cell receptors. Debromoaplysiatoxin inhibited the binding of these two substances as strongly as aplysiatoxin but did not increase malignant transformation or stimulate DNA synthesis. These results indicate that a slight change in the chemical structure of the hydrophilic region of aplysiatoxin affects its abilities to increase cell transformation and stimulate DNA synthesis and that the abilities of the tumor promoters to inhibit the binding of phorbol-12,13-dibutyrate and epidermal growth factor are dissociable from their abilities to increase cell transformation and stimulate DNA synthesis under some circumstances.

Two monomeric 32-kDa proteins, termed 32K-I (pI 5.8) and 32K-II (pI 5.1), were isolated from human placenta, which was solubilized by a Ca2+-chelator. Only 32K-I was associated with PLA2-inhibitory activity. CNBr peptide mapping indicated that 32K-I was distinct from 32K-II and two 36-kDa proteins, called calpactin I and II or lipocortin II and I, which have been shown to possess PLA2-inhibitory activity. 32K-I bound to PS in a Ca2+-dependent manner. 32K-I was detected in many tissues except brain, cardiac and skeletal muscle.

SOMATIC mutation has been considered a likely initiating step in chemical carcinogenesis1–3, chiefly because of the close correlation between the mutagenic and carcinogenic activity of various chemicals4–6. Study of cells from patients with disease predisposing to a high incidence of cancer, such as xeroderma pigmentosum and Fanconi's anaemia, has suggested that defective DNA repair is correlated with this predisposition to malignant transformation. When cells are transformed by 3-methylcholanthrene7, 4-nitroquinoline-1-oxide (4NQO)8, or X irradiation9–11, division is necessary soon after treatment to fix the transformation: if treated cells are unable to grow the transformation is not fixed. These results could be explained if DNA damage induced by a carcinogen is converted into a stable and replicable structural change only by means of DNA replication before the intervention of error-free repair mechanisms. The model of mutagenesis12–14 based on evidence obtained with micro-organisms suggests that errors introduced by an error-prone postreplication repair mechanism are the principal source of mutation. Furthermore, caffeine inhibits “postreplication repair” (presumably an error-prone mechanism) but not excision repair (presumably error-free) in rodent cells15–19. I now report that caffeine reduces the transformation frequency of mouse cells treated with 4NQO. This supports the somatic mutation theory of cell transformation.

Six variant clones isolated from a subclone of BALB/3T3-A31 clone were classified into three groups according to their different susceptibilities to cell transformation by ultraviolet light irradiation: highly susceptible, intermediately susceptible, and resistant. All variant clones showed similar susceptibility to cytotoxic effects induced by ultraviolet light.

We assessed the modulation of gene expression accompanying neoplastic transformation by computerized microdensitometry of autoradiographic patterns of [35S]-methionine-labeled polypeptides separated by two-dimensional polyacrylamide gel electrophoresis. Nearly 1000 polypeptide species of parent diploid human fibroblasts (KD strain) and clonally-derived malignant fibroblasts (HUT-14 strain) were compared. We found that the neoplastic HUT-14 fibroblasts express a mutation in one of the two functional beta-actin genes. In addition, of the 700 more-abundant polypeptides measured, 13 were lost and 14 new ones gained after this neoplastic transformation. We estimate that although 2% of fewer of the genes expressing abundant polypeptides were either activated or shut off, at least 32% were modulated quantitatively. A substrain of HUT-14--HUT-14T--shows increased tumorigenicity, producing larger, faster-growing fibrosarcomas in the nude mouse than does the present parent HUT-14 strain, and with fewer inoculated cells. This increase in tumorigenicity is accompanied by three subsequent changes in the mutant beta-actin polypeptide expression. A more variant mutant actin species is synthesized in HUT-14T, which differs from the original mutant polypeptide by (i) one additional negative net charge, (ii) a short half-life in the cell, (iii) a greatly diminished ability to incorporate into the detergent-resistant cytoskeleton, (iv) a decrease in affinity for deoxyribonuclease I (EC 3.1.21.1), and (v) a faster rate of synthesis. Our results suggest that a second-site mutation in the mutant beta-actin of HUT-14 was selected for during a subcloning step in the presence of 6-thioguanine before derivation of the HUT-14T substrain. This apparent mutation and two subsequent defective beta-actin expressions are accompanied by incremental increases of malignant potential.