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Diverse human tumors obtained directly from surgery or biopsy can grow at high frequency in 3-dimensional Gelfoam® histoculture for long periods of time and still maintain many of their in vivo properties. The in vivo properties maintained in vitro include 3-dimensional growth; maintenance of tissue organization and structure, such as changes associated with oncogenic transformation; retention of differentiated function; tumorigenicity; and growth of multiple types of cells from a single tumor.

Single-stranded DNA transcripts of rat type-C viruses prepared in the presence of actinomycin D, hybridized specifically to DNA of several rat cell cultures with no obvious qualitative or quantitative differences. Similar products prepared from a pseudo-type sarcoma virus with contributions from rat and mouse type-C viruses hybridized to both rat and mouse cellular DNA, while mouse viral transcripts did not hybridize to rat cell DNA. Viral RNA was detected in all rat cells by means of the rat viral DNA transcripts, with some differences between untreated low-passage cells and sister cultures treated with bromodeoxyuridine or bromodeoxyuridine and methylchol-anthrene. Cells treated with both compounds were previously shown to be transformed and tumorigenic, and these were distinguishable by kinetic analysis from the control cells.

Lung organ bits taken from full-term mice were explanted on the dermal surface of sterile, dead pigskin. The cells migrated onto the pigskin dermis and proliferated to form an organoid culture consisting of ductular structures separated by a matrix of epithelial cells. Cells within the ductular structures were ciliated, produced mucin, and exhibited the activities of nonspecific esterase and gamma-glutamyl transferase; therefore they were considered to be derived from bronchial epithelium. Cells forming the matrix possessed the activities of nonspecific esterase and alkaline phosphatase and contained lamellar structures typical of surfactant-producing pneumocyte Type II cells; therefore they were considered to be derived from alveolar precursor cells.

SUMMARY Susceptibility to chemically induced transformation changed as a rat embryo cell culture was passaged. For the first 35 to 60 passages, the cultures were diploid and resistant to transformation by chemical carcinogens. However, cultures infected with a murine leukemia virus were transformed by chemicals. For the next 60 passages, the cultures were heteroploid, but retained contact inhibition and were not tumorigenic. Even without addition of heterotypic viruses, these heteroploid cultures could be transformed by chemicals, but the endogenous rat C-type virus could be demonstrated in the transformed cultures. At higher passages, the rates of spontaneous transformation gradually increased so that the cultures could not be used for transformation studies. Chemically induced transformation of the stable heteroploid cell line (F1706) was manifested by an easy to read focal alteration. Initial observations based on these foci were confirmed by inoculating the morphologically altered cells into isogeneic newborn rats.

Mouse full-term embryonic lung tissue was cultured as organ bits using dead, sterile pigskin dermal collagen as a substrate. Explanted organ bits grew on the surface of, and into, the pigskin dermal collagen for at least 9 weeks after the initiation of culture. The outgrowth consisted of a thick cellular sheet containing various sizes of ductular structures within a cellular matrix that did not show any particular structure. Electron microscopic observation revealed that the larger ductular structures consisted largely of ciliated cells. The smaller ductular structure consisted largely of Type II pneumocytes containing lamellar bodies. The cellular matrix consisted of Type II pneumonocytes and other cell types including fibroblasts and macrophages in the early stage of cultivation. Macrophages invaded the pigskin dermal collagen. An intermediate cell type, which has never been observed in vivo, possessing both cilia and lamellar bodies was identified in the larger ductular structures. Upon comparison of the ultrastructure of the organoid in vitro cultures in pigskin with the original fetal lung, it was apparent that cytodifferentiation had occurred. The organoid components and structure of the cultured cells more closely resembled adult lung than the fetal lung used to initiate the cultures.

The literature on cell transformation by chemical carcinogens has been critically reviewed. This subject is highly relevant to carcinogenesis in vivo, because the phenotypic changes that are collectively referred to as cell transformation usually involve the acquisition of tumorigenicity on inoculation into suitable rodent hosts. The systems chosen for review fall into 3 categories: cell strains (cells with a limited lifespan); cell lines (cells with an unlimited lifespan); and oncogenic viral-chemical interactions involving cells (Fischer rat embryo cells expressing an endogenous retrovirus, mouse embryo cells expressing the AKR leukemia virus, chemical enhancement of a simian adenovirus, SA7 transformation of Syrian hamster or rat embryo cells). Of the entire literature reviewed, 117 papers have been accepted for data abstraction by pre-defined criteria; these include 41 references to cell strains, 40 in cell lines, and 38 in viral-chemical interactions including cells. Because different systems have been reviewed, it would be meaningless to group all the compounds. The overall summary of the systems is as follows (many compounds have been tested in more than one system and, hence, are duplicated in these totals). (Chart: see text) In general, there is a reasonably good correlation between the results of the cell transformation systems and in vivo carcinogenesis. However, the many deficiencies of the EPA Merged Carcinogen List preclude definitive comparisons. Moreover, a number of 'false negatives' were obtained in systems that did not employ external metabolic activation. Further validation of all systems is required, but it seems very probable that several cell transformation systems will become valuable in assaying (with reasonable time and cost) the carcinogenic potential of environmental chemicals.

Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10−8 M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10−6 M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10−8 M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method.

Myxoid chondrosarcoma is an uncommon neoplasm thought to be derived from mesenchymal chondrocytic cells. Although cytogenetic abnormalities have been reported in sarcomas, too few cases have been studied to determine the frequency of nonrandom chromosomal changes in mesenchymal tumors. In this article, we describe a chondrosarcoma with a nonrandom reciprocal translocation t(9;22)(q22;q11). The cellular homologue to the retrovirus transforming gene of simian sarcoma virus is located on chromosome #22, and its possible significance in this case is discussed.

The customary benzene extraction of airborne particulate matter collected in Los Angeles has previously been shown to yield carcinogenic material. The residue from the benzene extraction contains substances soluble in methanol, some of which are organic. This methanol extract and a number of its component fractions have been tested in rodent cell culture systems developed as assay methods for screening carcinogens. In one system a high-passage Fischer rat embryo cell line was used. In the other a low-passage cell line derived from NIH Swiss albino mouse embryos and infected with AKR leukemia virus was used. In both systems the methanol extract showed cell transformation activity approaching that of 3-methylcholanthrene. In the mouse cell system the activities of several fractions were also examined. The major inorganic component, ammonium nitrate, showed no activity. Neither the neutrals alone nor the recombined non-neutral fractions separately showed any activity, although they were active together. The basic materials separated by ion exchange were also active. The methanol extract combined with the conventional benzene extract showed much lower activity than either extract alone.

SummaryCell lines derived from adeno-virus-induced tumors or adenovirus-trans-formed cell lines clumped or retracted in 7.5 mM calcium or less, a characteristic generally not shown by cells derived from other virus-induced tumors or cells transformed by other viruses. Similarly, standard primary, diploid, and heteroploid cell cultures were not sensitive to 7.5 mM calcium. In support of these observations, the use of a low calcium medium facilitated the passaging of adenovirus-trans-formed cells in tissue culture; however, the use of a completely calcium-free medium resulted in a deficiency which caused detachment of the culture. The calcium effect may be useful as a marker to substantiate other evidence that a tumor was induced by adeno-virus or that a cell line was transformed by adenovirus.

Low-passage rat-embryo cells were not transformed by 3-methylcholanthrene or by 5-bromo-2′ deoxyuridine. However, prior treatment with bromodeoxyuridine, followed by treatment with methylcholanthrene, resulted in cell transformation about three subpassages after removal of the carcinogen. RNA-directed DNA polymerase activity could not be detected in either normal or transformed cells. However, gs-1 antigen specific for rat C-type RNA virus was detected in cultures derived from bromodeoxyuridine-treated cells. No gs-1 antigen for the C-type RNA virus was detected in cultures that had not been treated with bromodeoxyuridine during the 25 subpassages of these experiments. High-passage rat-embryo cells, derived from a different cell pool, were transformed by either methylcholanthrene or dimethylbenzanthracene without prior infection with an exogenous virus, and without prior treatment with bromodeoxyuridine, gs-1 antigen for C-type RNA virus was also detected in 4 of 4 randomly selected transformed cell lines; the gs-1 antigen was not detected in any of 4 nontransformed control cultures. Considering these and previously published findings, we conclude that the lowpassage cells cannot be transformed by methylcholanthrene because of powerful cellular controls over the endogenous virus. Bromodeoxyuridine triggers some expressions of the endogenous virus; thus, the bromodeoxyuridine-treated cells are more susceptible to the transforming effects of methylcholanthrene. High-passage rat cells do not maintain perfect control over expression of their endogenous virus; the cell cultures are susceptible to the transforming effects of chemical carcinogens.

A short, simple test involving interferon-mediated protection of cells in vitro from cytopathogenic effects produced by vesicular stomatitis virus or Sindbis virus has been developed to help in determining the genus of origin of cells. By using the observed pattern of protection of five cell types by five interferons, cells could be grouped as of primate, rabbit, rat, and mouse origins. The primate grouping resulted from bilateral cross-reactions between the human and monkey systems. An unexpected observation was that African green monkey interferon preparations protect both monkey and rat cells, but not the converse. Chromosomal analysis of the cell cultures confirmed the genus determined by the interferon test.

Monkey kidney cells tested in their first culture passage, 24 hours after their isolation from the animal host, required the same 13 amino acids for survival and growth as cell lines serially propagated in culture for years. Under the conditions of the present experiments, arginine, cystine, glutamine, histidine, and tyrosine proved necessary, over and above the 8 amino acids required for nitrogen balance in man. With the serially propagated lines, glutamic acid substituted for glutamine only at extremely high and non-physiological levels. In the monkey kidney cell cultures, however, glutamic acid and glutamine were interchangeable, mole for mole; and aspartic acid and asparagine were also effective as glutamine substitutes. Glycine was growth-stimulatory for monkey kidney cells in primary culture, and the cells grown in a glycine-deficient medium usually failed to survive subculture.

A number of normal rat cell cultures as well as cultures transformed spontaneously, by chemicals, and/or by oncogenic viruses were tested for responsiveness to the interferon inducer polyinosinic·polycytidylic acid, or to exogenous interferon. Responsiveness, or lack thereof, had no correlation with subculture passage number, infection with RNA leukemia virus, morphological transformation by oncogenic RNA or DNA viruses, chemical treatment, or the ability of these cells to produce tumors in isologous host animals. The data indicate that lack of response to interferon or to the inducer is neither a necessary prerequisite nor an absolute result of cellular transformation of rat cells.

CELLS derived from several animal species, including mouse1,2, hamster3 and rat4, have been transformed in vitro from the normal to the malignant state by diverse chemical carcinogens. In similar conditions, however, attempts to transform human cells have usually been unsuccessful and as far as we know, such transformation has been reported only once5. This was a case of two cell lines treated with urethane, obtained from siblings with von Recklinghausen's disease, a familial disorder transmitted by an autosomal dominant gene, and characterised by multiple fibromas with a high predisposition to malignant transformation in vivo6. Although morphologically altered foci of transformed cells were reported, there was the possibility that a few tumour cells in the original population had been selected for by urethane. Therefore we characterised in detail the urethane-treated and untreated cultures. We have found that human cells can indeed be chemically transformed in vitro.

RAT embryo cells infected with either CF-1 or Rauscher C-type RNA murine leukaemia virus, when treated with diethylnitrosamine (DENA), undergo morphological transformation and become aneuploid1. Untreated cells and cells treated with either virus or chemical alone do not transform. We describe here a similar effect of 3-methylcholanthrene (3 MC) on rat cells infected with Rauscher leukaemia virus.

Low passage Fischer rat embryo cultures, which are normally very resistant to transformation by 3-methylcholanthrene but are highly susceptible when chronically infected with the Rauscher murine leukemia virus, were completely protected from transformation by methylcholanthrene when treated with neutralizing antibody specific for the leukemia virus prior to and during treatment with methylcholanthrene. Sister cultures were not protected by neutralizing antibody specific for the B-tropic radiation leukemia virus. This demonstrates clearly a definite type specific role for Rauscher murine leukemia virus in the 2-methylcholanthrene transformation system in rat cells.

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.

Eight food dyes or commercial color mixtures certified for use in the United States were tested for their ability to transform in vitro a serial line of Fischer rat embryo cells previously reported to be a sensitive indicator of chemicals having carcinogenic potential. Malignant cell transformation was induced by a commercial mixture (G2024) of two of these dyes (Blue 1 and Yellow 5) and by Blue 2, Green 3 (one of two experiments) and Red 4. Food dyes Blue 1, Red 3, Yellow 5 and Yellow 6 did not induce cell transformation. One to 1.5 mg of each dye was injected into suckling LVG or Graffi hamsters which were monitored for tumor induction and/or death over a 330-day period. None of the non-transforming dyes (Blue 1, Red 3, Yellow 5, Yellow 6) or Green 3 induced a significant increase in tumor (mostly lymphoma) incidence or animal mortality. Three of the transforming dyes (Blue 2, Green 2024, Red 4) did increase tumor incidence and/or mortality in at least one strain of hamster. We conclude the the in vitro assay suggested that certain food dyes were carcinogens and that in vivo studies in hamsters supported this interpretation.

HIRATA, K., SHIRAMATSU, K., USUI, T., YOSHIDA, Y., FREEMAN, A.E. and HAYASAKA, H. Modulation of Fetal Mouse Liver Cells Cultured on a Pigskin Substrate. Tohoku J. exp. Med., 1983, 140, (1), 15-28-Mouse fetal liver cells cultured on a pigskin epidermal substrate grew for 7 weeks. Different enzymes and proteins, i.e. gamma-glutamyl transferase (GGT), nonspecific esterase (NE), lactic dehydrogenase (LDH), alpha-fetoprotein (AFP), and albumin were studied histochemically and/or biochemically. The activity of GGT was high at the beginning of culture and then decreased rapidly. The activities of NE and LDH were high during the culture. Release into the media and localization of AFP suggested active synthesis during the early stages. AFP levels gradually decreased and could be demonstrated only in trace amounts after 3-4 weeks of culture. On the other hand, the production of albumin was weakly evident early and became more and more evident after the second week in culture. Hydrocortisone modulated AFP and albumin production. The effect of hydrocortisone was to prolong expression of AFP and to reduce expression of albumin. Electron microscopic observations showed that the cultures consisted of organelle-rich parenchymal cells associated with the pigskin basement membrane by pseudopod-like structures. These results indicate that fetal mouse parenchymal cells were cultured and modulated on a pigskin epidermal substrate.

Cultured epithelial cells from human skin generally had 3- to 30-fold more hydrocarbon-metabolizing activity than fibroblasts from skin of the same donor. This activity was constant for up to 55 days in primary culture but was lost rapidly upon physical subdivision of the cultures. Treatment of primary mixed fibroblasts and epithelial cell cultures with methylcholanthrene, but not phenanthrene, led to development of actively growing fibroblastic cultures with many heteroploid cells. Unique marker chromosomes, stable over a number of cell population doublings, were identified in several of the heteroploid cell strains. Pure cultures of fibroblasts from the same donors did not undergo heteroploid conversion in response to methylcholanthrene. Spontaneously occurring heteroploidy in logarithmic phase human fibroblasts is a rare event; thus, heteroploid conversion may be a useful marker for chemical transformation of human cells. Because methylcholanthrene seems to have little transforming effect on human skin fibroblasts, human skin epithelial cells, because of their hydrocarbon-metabolizing activity, may serve to convert methylcholanthrene from a distal to an ultimate carcinogenic form.

Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing regeneration. After regeneration, the attachment to laminin returned to the low levels found in animals not subjected to partial hepatectomy but attachment to fibronectin remained high. Immunofluorescent staining of sections of normal liver with antilaminin revealed the presence of laminin in or adjacent to the walls of the bile ducts and blood vessels. After induction of regeneration by partial hepatectomy, increased amounts of laminin appeared in the sinusoidal areas. After carbon tetrachloride poisoning, staining for laminin was especially pronounced in the necrotic and postnecrotic areas around the central veins. This additional expression of laminin was transient. It reached a maximum around 5--6 days after the injury and then gradually disappeared. These findings show that laminin is an adhesive protein. The increase of laminin in regenerating liver and the adhesiveness of cells from such livers to laminin suggest a role for laminin in the maintenance of a proper tissue organization during liver regeneration.

In cases of extensive skin burns, there may not be sufficient viable skin to use as autografts. As a solution to this problem, we proposed that skin could be propagated in vitro, and the millions of cells thus grown could be used as autografts. The feasibility of this approach has been studied with rabbit skin. Under tissue culture conditions, small pieces of rabbit skin attach to supporting surfaces and proliferate to form layers of epithelial cells. A satisfactory supporting surface is the dermis of frozen porcine skin. With proper conditions, rabbit epithelial cell surface area can be expanded by a factor of 50 within 7 to 21 days.

Fetal mouse liver hepatocytes proliferate on a substrate of irradiated pigskin epidermis scored with scalpel blade slits to permit cell access to the basement membrane. At the time the cells are explanted, fetal genes, such as those responsible for production of alpha-fetoprotein (AFP) and gamma-glutamyltransferase (GGTase), are strongly expressed. The levels of GGTase decrease rapidly and become undetectable within 2 weeks. The levels of AFP decrease more gradually but become undetectable after 3-5 weeks in culture. As the AFP levels decrease, there is a concomitant increase in albumin production. Hydrocortisone prolongs production of AFP (for up to 8 weeks) but not of GGTase, and it decreases albumin production for up to 8 weeks. Once cells lose AFP expression, addition of hydrocortisone does not restart it. Based on these data, fetal mouse liver hepatocytes, cultured on pigskin, seem to be an excellent in vitro model for liver cell maturation.

In 129 of 140 attempts, human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin. Diploid epithelial cells grew selectively on the collagen bed; fibroblasts grew on the glass surfaces of the culture dishes. The cultures could be subdivided physically up to six times at a 1:2 split ratio, but at least 24 to 48 cell generations were produced over the months the cells could be carried. Much of the cell multiplication resulted in maturation into distinct basal, squamous, granular, and keratinized cell layers. The cultured cells were considered epithelial because of their shape, possession of intercellular bridges, desmosomes and tonofibrils, and because they formed maturating epithelium in vitro and upon transplantation back to the original human donor. As the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantitate cell growth. Multiplication of epilthelial cells, rather than migration, was indicated by mitotic figures in colchicine-treated cultures and by DNA synthesis.

We show that diverse human tumors obtained directly from surgery or biopsy can grow at high frequency in vitro for long periods of time and still maintain many of their in vivo properties. The in vivo properties maintained in vitro include three-dimensional growth; maintenance of tissue organization and structure, including changes associated with oncogenic transformation; retention of differentiated function; tumorigenicity; and the growth of multiple types of cells from a single tumor.

Seventy-five diploid human cell strains were subjected to a number of chemical carcinogens, including urethane and polycyclic hydrocarbons. In most cases, no visible morphological alterations were induced by any treatment. Development of morphologically altered foci was noticed in urethane-treated cultures derived from a patient with von Recklinghausen's disease. This disease is transmitted by an autosomal dominant gene, and has a high rate of spontaneous transformation of neurofibromas to neurofibrosarcomas. Attempts to isolate continuous cell lines from altered foci were successful in only two of several attempts. These continuous cell lines demonstrate altered morphology, loss of contact inhibition, accelerated growth rate, and have attained over 240 generations in a period of 140 weeks. Untreated control cultures became terminal by the 20th generation. Giemsa banding procedures showed that the chromosomal complement consisted of heteroploid human chromosomes. A second diploid cell strain derived from the above patient's sibling, also suffering from von Recklinghausen's disease, likewise was morphologically altered by urethane. Chemical transformation of human cells is difficult to induce; however, selection of genetically predisposed cells and prolonged, intermittent, and repeated chemical treatment may be important factors in achieving transformation.

ONCOGENIC human adenoviruses are readily divisible into two chief subgroups: A (types 12, 18, 31), and B (types 3, 7, 14, 16, 21 and probably 11) based on similar biological and biophysical properties1,2. Recently, Freeman et al.3 showed that adenovirus type 2, representative of a third subgroup of human adenoviruses, morphologically transformed rat embryo cells. The viruses of this subgroup, types 1, 2, 5 and 6, are similar in many biologic properties; they produce a partial haemagglutination pattern with rat erythrocytes, the agglutination being enhanced by the presence of heterotypic antibody4. Adenovirus type 4 also partially agglutinates rat cells, but in other respects, such as T antigen reactivity5, DNA–DNA homology6 and homology with tumour cell mRNA7, seems related to the B oncogenic subgroup. The rat cells transformed with adenovirus 2 contain an antigen which reacted in both complement-fixation (CF) and immunofluorescent tests with sera from hamsters inoculated with tumour extracts and with cells transformed by adenovirus 1- and 2-SV40 hybrid viruses3,8. These results suggested, as shown here, that other members of the adenovirus 1, 2, 5 and 6 subgroup would transform rat cells and that a common T antigen could be demonstrated. Evidence obtained by the fluorescent antibody method for a shared T antigen for adenoviruses 1 and 2 has already been reported8.

Adenovirus type 12 (Huie) inoculated into cultures of primary whole rat embryo produced foci of morphologically altered cells. The number and identification of these transformed areas was dependent upon the calcium concentration of the medium; more foci appeared in 0.1 m m than in 1.8 m m calcium. Cell lines derived from these inoculated cultures did not yield infectious virus, and also were similar to cell lines derived from adenovirus type 12-induced tumors with respect to morphology, presence of virus-specific tumor antigen, and oncogenicity. Dose-response curves revealed that transformation of rat embryo cells by adenovirus type 12 followed one-hit kinetics, and that approximately 7 × 10 5 infectious virus particles were required for one transformation event. Our results indicate that the transformation system described for adenovirus type 12 is reproducible, and that previous difficulties experienced in developing such a system may well be explained by the higher calcium concentration of the tissue culture media used.

Cell cultures of Syrian hamster embryo were treated for 7 days with selected chemicals. Certain cultures were morphologically transformed by three different chemical preparations and yielded cell lines that subsequently produced malignant tumors in hamsters. Although the cell lines were negative for infectious virus before inoculation into animals, hamster-specific C-type RNA virus was isolated from tumors or from cell lines derived from the tumors. Since infectious C-type viruses are usually not demonstrable in hamster tissues of normal or tumor origin, we conclude that the chemical treatment and activation of the viruses are related events.

Fischer rat embryo cells were treated with 3-methylcholanthrene before or after inoculation with Rauscher murine leukemia virus. Transformation was not observed in untreated control cultures, cultures given virus or 3-methyl-cholanthrene alone, or cultures treated first with 3-methylcholanthrene followed by inoculation with the virus after removal of the chemical. Transformation was dependent upon the presence of Rauscher murine leukemia virus at the time of chemical treatment.

The coverage of large surface area skin wounds with autograft skin is limited by the amount of available viable donor-site skin. A small amount of donor-site epithelium can be expanded many times over by cultivation in tissue culture on sheets of skin allografts or xenografts. Experiments performed in rabbits indicate that such autograft-allografts or autograft-xenografts can be transplanted to large surface area wounds with essentially complete epithelial coverage of the wounds within two to three weeks, even with wound surface area coverage 50 times the surface area of donor-site skin.