Multiple mechanisms for the carcinogenic effects of asbestos and other mineral fibers.

Asbestos and other mineral fibers are carcinogenic to humans and animals but differ from many carcinogens in that they do not induce gene mutations. An understanding of these interesting human carcinogens, therefore, is an important problem in cancer research. Asbestos and other fibers induce predominantly two types of cancers: mesotheliomas and bronchogenic carcinomas. Fiber size is an important factor in the carcinogenic activity of these substances as has been shown for mesothelioma induction. For bronchogenic carcinomas, but not for mesotheliomas, a synergistic effect of asbestos exposure and cigarette smoke has been observed in humans. The mechanisms by which fibers alone versus fibers in concert with other carcinogens induce cancers are probably distinct. In addition to fiber dimensions, fiber durability and surface properties of fibers are important properties affecting carcinogenicity. Evidence exists that asbestos is a complete carcinogen, an initiator and a promoter. Multiple mechanisms must be operative to explain the diverse effects of mineral fibers. Although asbestos is inactive as a gene mutagen, there is now clear evidence that it induces chromosomal mutations (aneuploidy and aberrations) in a wide variety of mammalian cells including mesothelial cells. Asbestos also induces transformation of cells in culture including mesothelial cells and fibroblasts. A mechanism for cell transformation, which is dependent on fiber dimension, has been proposed. The fibers are phagocytized by the cells and accumulate in the perinuclear region of the cells. When the cell undergoes mitosis, the physical presence of the fibers interferes with chromosome segregation and results in anaphase abnormalities. The transformed cells show aneuploidy and other chromosome abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinogen risk assessment.

A molecular biological rationale for the linear nonthreshold dose-response pattern for carcinogenesis is presented based on the mutagenic activation of oncogens as the basis of initiation. The approach assumes that the linear nonthreshold dose pattern at very low doses applies only to tissues that are promoted by intrinsic and extrinsic agents other than the one being modeled, and that risk is characterized on a relative rather than absolute basis in terms of aggregate tumor response.

Biological effects of asbestos fibres on rat lung maintained in vitro.

The in vitro systems used to determine whether asbestos acts as an initiator or as a promoter have failed to give definitive answers. We studied the effect of chrysotile and crocidolite in an initiation-promotion model on the Fischer rat embryo lung. Two assay systems were used in succession: organ culture of the lung cultured for 24 days and epithelial cell culture derived from treated or untreated explants cultured for 25 passages. Apart from the control groups, three major groups were analysed: (1) fibres with complete carcinogenic potency: explants and/or cells treated with fibres alone; (2) fibres with initiating potency; short treatment with fibres, followed by treatment with the classical promoter TPA; (3) fibres with promoting potency: short benzo[a]pyrene treatment followed by treatment with the fibres. In organ culture, fibres alone induce only cytotoxic lesions; in the 'fibres with promoting potency' group, precancerous lesions were observed. In epithelial cell culture, several transformation criteria are analysed. Our results with the cell system confirm that fibres act as a promoter, but also as a complete carcinogen. However, for equal doses, crocidolite needs a longer treatment time than chrysotile. These different assays failed to demonstrate any initiating activity of the fibres. The use of organ and cell culture in succession makes it possible to demonstrate the in vitro promoting effect of chrysotile and crocidolite.

Continuous-wave ultrasound and neoplastic transformation in vitro.

C3H/10T1/2 cells in suspension were assayed using an initiation-promotion protocol for neoplastic transformation induced by continuous-wave ultrasound. Cells were insonated at 1.765 MHz for 40 min. Two ultrasonic intensities were used: 1.3 and 2.6 W/cm2 spatial average. The first intensity was found to be noncytotoxic; the second resulted in immediate lysis of 20% of the cells, followed by the clonogenic survival of 64% of the remaining cells. Ultrasound was delivered alone or in combination with X-rays (2 Gy, 240 kVp given before ultrasound), and/or 12-O-tetradecanoyl-phorbol-13-acetate (TPA, 0.1 microgram/ml post-irradiation). Under all treatment conditions, there was no effect of ultrasound on transformation at the 95% confidence level.

Chemical carcinogen Epstein-Barr virus (EBV) synergism: EBV genome amplification and site-specific mutation during transformation.

When treated with chemical carcinogens, peripheral blood lymphocytes (PBLs) from normal adults or patients with acquired immunodeficiency syndrome (AIDS) were more readily transformed than non-treated PBLs into immortalized lymphoblastoid cell lines (LCLs) by the Epstein-Barr virus (EBV, FF41-1). Chemical carcinogens also enhanced spontaneous transformation in both PBL populations. Co-cultivation of lethally ultraviolet (UV)-irradiated uninfected PBLs with EBV-infected PBLs resulted in a dose-related enhancement of transformation, providing evidence that enhancement is in part mediated by carcinogen-induced diffusible trans-acting factors. Analysis of the EcoRI J region of resident EBV genomes showed that LCLs established from carcinogen-treated PBLs frequently had EBV genome amplification higher than that seen in controls. Carcinogen-induced EBV genome amplification was shown to be dose-related. The organization of the terminal region of the EBV genome was analyzed in LCLs derived from carcinogen-treated PBLs and compared to that of FF41-1 and B95-8. LCLs established from N-methyl-N'-nitroso-nitrosoguanidine (MNNG)- and aflatoxin B1-treated PBLs frequently had 2 circular forms of the EBV genome and several linear forms varying by numbers of terminal repeats (TRs) at the 3' and 5' end. This is in contrast to the single episomal circular and linear form of the EBV genome found in the parent FF41-1 and single episomal circular form in control FF41-1-carrying LCLs. Linear forms of the EBV genome were only found in FF41-1-transformed LCLs derived from carcinogen-treated PBLs and were associated with the production of unusually large amounts of infectious EBV. These results demonstrate that chemical carcinogen-mediated enhancement of transformation is accompanied by quantitative and qualitative alterations in the physical structure, organization and expression of the EBV genome which may in turn be the result of a carcinogen-induced cellular SOS response.

Modification of fibrous Oregon erionite and its effects on in vitro activity.

A sample of erionite, a fibrous zeolite, was modified by milling to reduce the number and length of the fibres and by extraction with cyclohexane. The in vitro activities of this mineral were found to depend on the presence of long fibres. The erionite contained fewer of these fibres than the UICC asbestos samples but, unlike these materials, erionite can cause the transformation of C3H 10T1/2 cells. Erionite did not increase the activities of benzo[a]pyrene in this cell transformation assay. The cytotoxic activities of both asbestos and erionite have a similar dependence on the number of long fibres. Extraction with cyclohexane did not affect the activity of erionite.

Transforming DNA sequences of human hepatocellular carcinomas, their distribution and relationship with hepatitis B virus sequence in human hepatomas.

Several related human transforming DNA sequences, hhc, and a putative normal liver homologue, c-hhc, have been molecularly cloned from the genomic DNAs of individual African and Asian hepatomas and from normal liver respectively. hhcM (Mahlavu) and hhcK3 (Korean), but not c-hhc, transformed NIH3T3 cells in DNA-mediated gene transfer assays. Transformed cells were found tumorigenic in athymic NIH Swiss nu/nu mice. In view of recent epidemiological studies implicating hepatitis B virus (HBV) infection early in life as causative for the eventual development of primary hepatocellular carcinoma in humans in Southeast Asia, the Far-East, and certain areas of Africa, we hereby analyzed the relationship between these hhcs and HBV in a survey of 20 hepatomas for DNA sequences homologous to hhcM and HBV by sequential hybridizations against [32p]hhcM and [32p]HBV probes. hhcM related DNA sequence were found highly amplified in 80% of the 20 hepatomas but HBV DNA sequence was rare or low. hhcM lends itself as a marker for human hepatomas. However, overall results indicated that patients with integrated HBV DNA sequences showed high copy number of hhcM sequence. Furthermore, EcoR1-restricted hepatoma DNAs showed that HBV and hhcM DNA sequences resided at different fragments in hepatomas. Our results suggest that HBV contributes to hepatocarcinogenesis probably via an activation mechanism involving possibly an integration or transient interaction of HBV DNA with hepatocyte DNA sequences, leading to recombination and eventual amplifications of the hhcM sequence in Mahlavu.

Truncation of the human EGF receptor leads to differential transforming potentials in primary avian fibroblasts and erythroblasts.

The transforming capacity of the normal and mutant human EGF receptor (EGFR) was investigated in primary chicken cells. In fibroblasts, both N- and C-terminal truncations resulted in a weak, additive oncogenic activity. However, not even double truncations caused a v-erbB-like phenotype. Upon EGF-binding, on the other hand, both normal and C-terminally truncated EGFRs resembled v-erbB in their fibroblast transforming potential. In erythroblasts, N-terminal truncation was sufficient to induce constitutive self-renewal, which was enhanced by deletion of 32 C-terminal amino acids but abolished by a larger truncation of 202 amino acids. In contrast to the normal EGFR, the receptor lacking 32 C-terminal amino acids resembled v-erbB in conferring erythropoietin independence for spontaneous differentiation to the transformed erythroblasts. Our results indicate that the C-terminal domain of the EGFR is non-essential in fibroblast transformation, but seems to be crucial for both self renewal induction and specificity of receptor function in erythroblasts.

A threshold effect in the induction of tumorigenicity of an established human cell line by v-mos.

The nontumorigenic immortal human cell line, SV80, was transfected with the v-mos gene to assess the gene's effect on tumorigenicity of cultured human cells. Two classes of cells, each containing functional v-mos, were obtained. The first class contained low levels of v-mos RNA, was morphologically transformed, but was nontumorigenic in nude mice. The second was also morphologically transformed, but contained high levels of v-mos RNA and was tumorigenic. The results indicate that SV80 cells behave similarly to murine fibroblasts in their response to v-mos in that they can be rendered tumorigenic by the viral oncogene. However, tumorigenicity was effected through a mechanism which involves different threshold doses for morphologic and tumorigenic transformation.

Skin types in dysplastic nevus syndrome.

In order to determine if individuals with dysplastic nevi (DN) are relatively more sun-sensitive than controls who do not have DN, the sun-reactivity skin types (based on the Harvard classification) were determined in these two groups. Compared with controls, sun-sensitive types were significantly overrepresented in the DN group. This is consistent with the hypothesis that the fundamental defect in the dysplastic nevus syndrome is the genetically unstable melanocyte, which is susceptible to neoplastic transformation induced by sunlight.

Efficient malignant transformation of rat embryo fibroblasts by genomic DNA from Walker carcinoma cells.

DNA isolated from Walker carcinoma ascites cells was transfected into primary rat embryo fibroblasts (REF), selecting transformed cells by growth in soft agar after prolonged propagation in monolayer. Both high molecular weight genomic DNA and a partially purified mitochondrial DNA fraction were able to transform REF with high efficiency, whereas pure mitochondrial DNA failed to elicit a transformed phenotype. Hybridization experiments showed that the mitochondrial DNA fraction contained DNA species of presumably extramitochondrial origin. Colonies were cloned into morphologically transformed, foci-forming, immortalized cell lines, showing different degrees of chromosomal alterations, tumorigenicity, and production of cell growth factors. These results indicate that although REF are refractory to genomic neoplastic DNA or to single cloned oncogenes in the absence of enhancers, they can be efficiently transformed by chromosomal DNA from a highly malignant tumor under conditions selecting against the remaining normal cells.

Expression of the control elements of BK and SV40 viruses in human cells exhibiting different transformed phenotypes.

Transformation of primary human embryonic kidney (HEK) cells as selected by either focus assay or growth in soft agar after their transfection with BK virus (BKV) DNA alone or BKV DNA plus the activated form of human Ha-ras oncogene has been previously reported (A. Pater and M. M. Pater, J. Virol. 58, 680-683 (1986]. In order to examine the expression of the regulatory elements of papovaviruses in each of the transformed phenotypes, chloramphenical acetyltransferase (CAT) plasmids under the control of BK or SV40 early promotor were used in transient assays. The expression of CAT, as driven by either SV40 or BK early promoters, was approximately 100-fold higher in HEK cells transformed by BKV DNA plus Ha-ras oncogene than in cells transformed only by BKV DNA. The higher CAT expression was not due to high levels of plasmid replication in these cells. Time course studies revealed that the higher CAT activity could be explained largely by greater plasmid uptake and stability in BK plus ras-transformed cells.

Characterization of the transformation properties of Shope fibroma virus.

To investigate if Shope fibroma virus (SFV), a leporipoxvirus that induces benign tumors in adult rabbits, can trigger the second step of carcinogenesis in vitro or malignant transformation, an already immortalized rabbit cell line (SIRC) was inoculated with ultraviolet-irradiated virus. The resulting cell transformants displayed the characteristic properties of the malignant phenotype: lack of infectious particles, low serum requirement, high efficiency of cloning, resistance to superinfection, presence of viral DNA sequences in the nucleus, expression of viral proteins and induction of tumors in rabbits. However, this transformation was not stable since in all cell lines studied, a loss of the malignant phenotype was recorded close to the 50th passage. To assess the oncogenic potential of SFV, NIH 3T3 cells were transfected with SFV DNA. The results of these experiments indicate that SFV DNA can induce the formation of foci in certain NIH 3T3 cell lines. Taken together these results support the notion that SFV can elicit the transformation of cells in vitro.

Activation of pp60c-src transforming potential by mutations altering the structure of an amino terminal domain containing residues 90-95.

The overexpression of the c-src gene product, pp60c-src, in avian and rodent embryo cells is not sufficient to induce cellular transformation. In this study we report that structural alterations within an amino terminal domain of pp60c-src, the exon 3 domain (residues 84-115) activate the oncogenic potential of the c-src gene product. Site-directed mutagenesis of the c-src gene was used to generate c-src variants encoding pp60c-src proteins with the following amino acid alterations: tyr 90 to phe (pm90F); tyr 92 to phe (pm92F); arg 95 to either trp, lys, glu or gln (pm95W, 95K, 95E and 95Q, respectively), and deletion of residues 92-95 (dl92). C-src variants encoding proteins with the alteration of arg 95 to trp, glu, or lys, or containing the deletion of residues 92-95, induced alterations in cell morphology and promoted growth in soft agar as well as changes in glucose transport and in vivo tyrosine phosphorylation of cellular proteins (including calpactin I heavy chain, p36). Analysis of in vivo phosphorylation of the transforming variant src proteins revealed little detectable alteration in the phosphorylation of tyr 527, a putative site of tyrosine kinase regulation. Our results suggest that structural alterations within a domain distal to the catalytic (kinase) domain activate pp60c-src kinase activity and, concomitantly, oncogenic potential. Furthermore, we suggest that the exon 3 domain of pp60c-src may contribute to the regulation and/or substrate specificity of the c-src protein.

Infección crónica por el virus de hepatitis b (VHB) y desarrollo de carcinoma primario del higado (CPH) / Chronic infecction by hepatitis B virus and development of primary carcinoma of the liver

Una de las alteraciones más importantes consecutivas a la infección con el VHB en el hombre, es la infección crónica y el estado de portador. En los últimos 15 años se han publicado estudios que sugieren la asociación entre la infección crónica con el VHB y el CPH, claculándose que en el mundo se diagnostican 250,000 casos nuevos de CPH cada año. Esta observación se apoya al conocerse las bases moleculares de la lesión hepática por el VHB que han permitido estudiar la expresión y la replicación del virus, el análisis del DNA viral, el RNA viral y toda la estructura proteíca del virus. Al utilizar técnicas de biología molecular se aumentó el conocimiento del virus B, particularmente en lo que se refiere al desorollo de hepatitis crónica, al estado de portador y parcialmente a los eventos que ocurren durante la progresión a hepatocarcinoma. Recientemente se ha demostrado la integración del DNA del VHB en el genoma de los hepatocitos que desarrollan transformación maligna. Esta integración del DNA del VHB se ha encontrado también con un patrón idéntico en regiones cercanas al tumor, hecho que para algunos autores sugiere que la integración de la secuencia del virus B en el DNA del huésped preced de hecho a la oncogénesis hepática. El tiempo exacto en el que ocurre la integración no se conoce, sin embargo, estudios epidemiológicos sugieren que se requieren más de 10 años, quizás entre 20 y 30 años para que se desarrolle el CPH; por lo tanto, los individuos portadores crónicos del VHB desde los primeros años de la vida, son los que están en mayor riesgo de desarrollar hepatocarcinoma. Los factores etiológicos que participan en el desarrollo del CPH son los siguientes: a.- Persistencia del virus de la hepatitis B (estado de portador). b.-Edad y sexo. c.- Duración de la infección. d.- Susceptibilidad genética. e.- Condiciones ecológicas favorables. f.- Otros factores

A theory of carcinogenesis based on an analysis of the effects of carcinogens.

Carcinogenic stimuli appear to act on target cells (and their daughters) by one or more of three mechanisms. The first is by oxidation of membrane component molecules on the extracellular surfaces of their plasma membranes. The second is by chronic and continuous impingement of electrons on the extracellular surfaces of their plasma membranes and the third is by relocation of predominantly basic molecules to the cytoplasmic surfaces of their plasma membranes. This latter effect in turn causes electrostatic attraction of image charged acidic molecules to the extracellular surfaces to balance the transmembrane charge of the target cells. Each of the above mechanisms results in a condition of increased electronegativity of the extracellular surfaces of plasma membranes of the target cells and their daughters. A theory of transformation is advanced based on the above related modes of action and it is used to explain some previously unexplainable properties of tumors.

Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis of NIH 3T3 cells.

The HER2 gene encodes a cell-surface glycoprotein with extensive homology to the epidermal growth factor receptor. Recently it was found to be amplified in about 30% of primary human breast malignancies. In experiments designed to assess the role of the HER2 gene in oncogenesis, we found that overexpression of unaltered HER2 coding sequences in NIH 3T3 cells resulted in cellular transformation and tumorigenesis.

Growth in culture and tumorigenicity after transfection with the ras oncogene of liver epithelial cells from carcinogen-treated rats.

Two epithelial cell lines designated LE/2 and LE/6 were established from cells isolated by centrifugal elutriation from the livers of carcinogen-treated rats. Both cell lines exhibit some characteristics of fetal liver cells, such as the expression of the 2.3-kilobase alpha-fetoprotein mRNA, aldolase A, and lactate dehydrogenases 4 and 5. Primary cultures contain gamma-glutamyl transferase-positive cells which do not proliferate in vitro. After the first passage, the LE/2 and LE/6 cell lines are uniformly gamma-glutamyl transferase negative. Neither cell line is transformed as assayed by morphology, anchorage-independent growth, or tumor formation in nude mice. By the 50th passage, LE/6 cells form numerous colonies in soft agar in the presence of epidermal growth factor, while no colonies grow in medium lacking this growth factor. Clonal cell populations derived from five epidermal growth factor-induced soft agar colonies were not tumorigenic in nude mice. This indicates that, although epidermal growth factor-responsive late passage cells had acquired some of the phenotypic properties commonly associated with tumor cells, these cells were not fully transformed. Transformation of LE/6 cells was accomplished by transfection of the rasH oncogene (EJ). Subcutaneous inoculation of rasH (EJ)-transfected LE/6 cells produced tumors at the site of injection with histological features of moderate to well-differentiated trabecular hepatocellular carcinomas. Tumor cell lines derived from the nude mouse tumors are gamma-glutamyl transferase positive and express alpha-fetoprotein mRNA. One clonal cell line expresses both alpha-fetoprotein and albumin mRNA. These results show that nonparenchymal liver epithelial cells transfected with an activated oncogene can give rise to differentiated hepatocellular tumors similar to those induced in livers of rats fed a carcinogenic diet.

Oncogene-mediated multistep transformation of C3H10T1/2 cells.

We have examined the response of the mouse embryonic cell line C3H10T1/2 to transfection with the activated human c-H-ras oncogene and the gag-myc oncogene from avian myelocytomatosis virus 29. C3H10T1/2 cells are not morphologically transformed following transfection with the gag-myc oncogene. A low level of focus formation is observed following transfection of the c-H-ras oncogene. When C3H10T1/2 cells are cotransfected with the ras and myc oncogenes, focus formation is increased by an average of 13 fold. In addition, C3H10T1/2 ras/myc foci have a distinct, transformed morphology which correlates with an increased potential for anchorage-independent growth. Although morphological transformation in this system is largely a function of ras oncogene expression, our studies demonstrate that it is potentiated by the presence of a functional gag-myc protein. Oncogene-mediated multistep transformation, which was first described in primary embryo cultures, is not a general property of established cell lines. The C3H10T1/2 cell line is an exception and provides a model system in which partially transformed phenotypes, in a progression toward malignant transformation, can be isolated and studied.