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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.

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.

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

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.

The modulation of gene expression accompanying neoplastic transformation has been assessed by computerized microdensitometry or 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. HUT-14 fibroblasts express a mutation in one of the two functional beta-actin genes and possess properties that distinguish them as neoplastic cells. Of the 700 more abundant polypeptides measured, 13 were lost and 14 were gained following this neoplastic transformation. It is estimated that less than or equal to 2% of the genes expressing abundant polypeptides were either activated or shut off, but at least 32% were modulated quantitatively as a consequence of this neoplastic transformation. Classes of "highly variable" and "marginally variable" polypeptides were assigned. Among the "highly variable" polypeptides, two related species barely detectable in KD parental cells were synthesized at a 25-31-fold higher rate in the transformed cells, and the cell-associated and extracellular matrix forms of fibronectin were each diminished by greater than 90%. Principles emerging from this study may form a basis for interpretation of the role of individual genes in the expression of neoplastic characteristics of HUT-14 cells.

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.

A clone of spontaneously transformed Chinese hamster lung cells was exposed to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), and six heat-sensitive and three cold-sensitive mutants were isolated after selection for inability to form colonies in soft agar at 39.5 degrees C and 34.5 degrees C, respectively. The heat-sensitive mutants had growth characteristics of transformed cells at 34.5 degrees C, but exhibited a normal phenotype at 39.5 degrees C. By contrast, cold-sensitive mutants displayed the characteristics of the normal cells at 34.5 degrees C and converted to a transformed phenotype at 39.5 degrees C. Transformed parent cells exhibited no obvious temperature-dependent properties. Temperature shift experiments showed that the colony-forming ability of both types of mutants was fully reversible. All of the mutants were able to grow well at both permissive and nonpermissive temperatures when grown on the surface of plastic dishes. Such mutants will be useful in analysis of factors involved in the expression of the transformed state or the maintenance of the nontransformed state.

The induction of DNA synthesis in quiescent, density-arrested Balb/c 3T3 cells is known to be controlled by the sequential action of at least two functionally distinct sets of growth factors, so-called "competence factors" and "progression factors." Here we examined this induction pathway in Balb/c 3T3 A31-I variants, which showed differential susceptibilities to radiation- and chemical-induced neoplastic transformation despite their similar susceptibilities to radiation- or chemical-induced cell killing and mutagenesis. DNA synthesis was acquired only with the exposure to progression factors in a highly susceptible cell variant (A31-1-13) whereas both competence factors and progression factors were required for a less susceptible cell variant (A31-I-1). The competent state constitutively produced by an autologous mechanism in the highly transformation-susceptible A31-I-13 cells suggests the existence of an endogenous promoter that acts for the expression of the transformed phenotype in an autocrine fashion when the cells have been initiated by radiation or chemical carcinogens. The growth factor requirements acting as a determining factor for susceptibilities to transformation are discussed.

In order to obtain more information on the in vitro transformation of human cells, a human fetal tracheal epithelial cell line (FHET16/5) was exposed for a long time to diethylnitrosamine (DEN). In 20 passages, this cell line (diploid, male) maintained strong immunohistochemical reactivity for carcino-embryonic antigen and wool merokeratin; it was negative for vimentin. The cells contained PAS-positive mucous substances and ultrastructurally were found to have desmosome-like attachments. Treatment of the cells was with 0.3% dimethyl sulphoxide (DMSO), or DMSO with 150, 450, 1000 or 2000 micrograms/ml of DEN. It was started at the ninth passage and continued for six passages over 9 weeks for the control (DMSO) and the three lowest control doses of DEN, and for three passages over 9 weeks for the 2000 micrograms/ml DEN group. Cells grown for 13 days after the end of treatment were plated in soft agar and injected subcutaneously in nude mice. The frequency of anchorage-independent colonies grown in soft agar was directly related to DEN dose. Colony-forming efficiency, as an expression of toxic effect, was also dose dependent. Autoradiographically detected unscheduled DNA synthesis indicated an association between anchorage-independent transformation and DNA alterations induced by DEN. Cells injected into nude mice did not produce tumours during a 6-month period, but invasiveness was observed when cells from the 2000 micrograms/ml DEN group were transplanted on the dermis of cultured chick embryo skin. The results indicate that DEN causes anchorage-independent transformation accompanied by unscheduled DNA synthesis in a fetal human tracheal epithelial cell line.

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.

To analyze the mechanism of neoplastic transformation of rodent diploid cells by ras and myc oncogenes, human EJ c-Ha-ras and mouse c-myc second and third exons promoted by SV40 promoter were connected to pSV2neo and pSV2gpt, respectively. Mouse and rat primary fetal cells cotransfected with both genes formed transformed and nontransformed colonies in a medium containing G418 and myc ophenolic acid (MPA). The proportion of transformed colonies in the total G418/MPA-resistant colonies decreased dependent on the stage of the gestation period of rat fetuses from which primary cells had been obtained. Analysis of randomly isolated colonies showed that the transformed colonies had a high copy number and high amount of expression of the introduced genes, were anchorage independent, and were tumorigenic in nude mice. On the other hand, the nontransformed colonies had a low copy number, low amount of expression, and no tumorigenicity. This contrast indicated not only that the activated Ha-ras and myc oncogenes had been integrated, but also that the amplification or overexpression (or both) of these genes was required for the rodent diploid cells to be transformed. We conclude that early-stage rat fetal cells might have endogenous factors that promote cell transformation. Alternatively, late-stage cells might have factors that suppress cell transformation by activated Ha-ras and myc oncogenes.

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.

A31-714 cells, a subclone derived from BALB/3T3, did not produce transformed foci when kept under culture conditions of density-dependent inhibition after treatment with 4NQO in either the growing or the non-growing state. The transformation frequency increased to nearly maximum level only when the cells had undergone more than four cell generations after 4NQO-treatment. Cells which were first kept in a non-growing state after 4NQO-treatment, rapidly lost the ability to exhibit transformation even when they were subsequently returned to a growing state. On the other hand, the cells which were allowed one cell replication soon after 4NQO-treatment retained their ability to produce transformed foci even after being kept in the non-growing state for as long as 6 days thereafter. These results suggest that one cell generation is required for the fixation of transformation, and more than four cell generations are required for the expression of the transformed state.

A31–714, a subclone isolated from clone A31 of the BALB/3T3 line showed a high degree of contact inhibition and an extremely low incidence of spontaneous transformation. Treatment of this subclone with 4-nitroquinoline-1-oxide, 3-methylcholanthrene, benzo(a)pyrene or N-methyl-N'-nitro-N-nitrosoguanidine produced foci of multilayered growth on the background of contact-inhibited monolayer within 2 to 4 weeks. In this system the transformation frequency can be determined quantitatively on either an inoculated cell basis or a surviving cell basis. The transformation frequency increased with the concentration of carcinogens within a certain range, and was affected by the duration of treatment and the cell density at the time of treatment. Treatment with DMSO alone or with the non-carcinogenic substances, 4-nitroquinoline, 4-aminoquinoline-1-oxide, phenanthrene and pyrene, did not cause any transformation. The cytotoxic effects of carcinogens were not directly correlated with the transformation frequencies. All transformed cell lines derived from each focus showed characteristics known as the indices of malignant transformation, such as a criss-cross pattern or piling up of cells, a high saturation density and the ability to grow progressively in soft agar. When the transformed cells were injected into the skin of the back of newborn or adult mice at a dose of 106 cells, fibrosarcomas were produced at the site of injection after 3–6 weeks. Neither untreated A31–714 cells nor morphologically untransformed cells in cultures with foci produced tumors on injection at a dose of 107 cells. In general, transformed cells had a reduced cloning efficiency, and almost the same susceptibility to the cytotoxic effects of carcinogens as untransformed cells, though these properties varied in different lines.

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.

HUT 14 is a cloned transformed cell line derived from normal diploid human KD fibroblasts. HUT 14 cells have an altered actin phenotype. In addition to the two nonmuscle actins beta and gamma, also present in the parent KD cells, they show the stable expression of a novel actin species (Ax-actin). Amino acid sequence analysis has been used to identify the three actins of HUT 14 cells. beta- and gamma-actins are identified as normal mammalian nonmuscle actins whereas Ax-actin is characterized as a beta-actin mutant revealing a single amino acid substitution at position 244. The results obtained are compatible with a simple mutational event involving a point mutation in one of the two beta-nonmuscle actin genes assumed to be present in proliferating human diploid fibroblasts. Certain emerging principles of nonmuscle actin gene expression in higher vertebrates are discussed.

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.

When highly transformable BALB 3T3-A31 clone 1–13 cells are exposed to benzo(a)pyrene for various lengths of time, only one type of carcinogen-DNA adduct is detected. No minor adducts were found as reported in other cell systems. High pressure liquid chromatography identified this persistent adduct as 10-trans-7R-benzo(a)pyrene diolepoxide I-deoxyguanosine. Formation of benzo(a)pyrene-deoxyguanosine adduct, therefore, appears sufficient to initiate the transformation process induced by benzo(a)pyrene in 1–13 cells.

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.