methylnitronitrosoguanidine and Tracheal-Neoplasms

The effects of sodium arsenite (As) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on epithelia of human fetal trachea and bronchiolar epithelia of human fetal lung were studied by using organ-cultured explants. In epithelium of human fetal trachea, 34 microM MNNG induced hyperplasia, metaplasia, and dysplasia; 1 microM As induced hyperplasia; and 3-9 microM As induced hyperplasia and cellular atypia. In glandular epithelium of human fetal trachea, 34 microM MNNG induced hyperplasia and metaplasia; 1 microM As did not induce obvious changes; and 3-9 microM As induced hyperplasia and epidermoid metaplasia with nuclear atypia. In bronchiolar epithelium of human fetal lung, the induction of dysplasia was observed for 1 microM As. Arsenic-induced preneoplastic lesions support the conclusion of epidemiological studies that arsenic is carcinogenic to human lung.

Topics: Arsenites; Bronchi; Epithelium; Fetus; Humans; Hyperplasia; Lung; Lung Neoplasms; Methylnitronitrosoguanidine; Organ Culture Techniques; Precancerous Conditions; Sodium Compounds; Trachea; Tracheal Neoplasms

The transforming potency of ozone for rat tracheal epithelial (RTE) cells exposed in vivo or in vitro was determined. RTE cells isolated from rats exposed to ozone (0, 0.14, 0.6, or 1.2 ppm, 6 hr/day, 5 days/week for 1, 2, or 4 weeks) showed no increase in the frequency of preneoplastic transformation compared to cells isolated from unexposed rats, although ozone-induced morphologic changes were observed in exposed tracheas. In contrast, preneoplastic variants of RTE cells were induced by multiple, but not single, exposures of RTE cells to ozone in culture. RTE cells exposed biweekly to ozone (approximately 0.7 ppm for 40 min, nine total exposures) had approximately twofold increases in the frequency of preneoplastic transformation compared to that of concurrent controls exposed to air. Single, 40-min exposures to ozone (approximately 1 or approximately 10 ppm) did not induce preneoplastic variants. However, single, 40-min exposures of RTE cells to approximately 10 ppm ozone did result in approximately 40% decreases in colony-forming efficiency. In addition, single, 40-min exposures of RTE cells to approximately 1 ppm ozone reduced the transforming potency of a subsequent exposure to the direct-acting chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When multiple ozone exposures followed exposure to MNNG (approximately 0.7 ppm ozone for 40 min, nine biweekly exposures), an additive (or possibly a multiplicative) effect of ozone on MNNG-induced preneoplastic transformation was seen. These results demonstrate that ozone can, under some conditions, induce preneoplastic variants of RTE cells. In addition, depending on the sequence or combinations of exposures, ozone can reduce or, possibly, increase, the transforming potency of the carcinogen MNNG for rat tracheal cells in culture.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Drug Interactions; Male; Methylnitronitrosoguanidine; Ozone; Precancerous Conditions; Rats; Rats, Inbred F344; Trachea; Tracheal Neoplasms

The effects of As2O3, MNNG and B(a)P on epithelia of human fetal tracheae and rat tracheae in organ culture were studied. In human fetal trachea, a small dose of arsenic (1 mumol As) induced hyperplasia of the epithelium; 3-9 mumol As induced hyperplasia and cellular atypia in the epithelium, and hyperplasia and squamous metaplasia in the adenoepithelium. Similar effects were not observed in rat tracheae. MNNG and B(a)P induced hyperplasia, squamous metaplasia and dysplasia in human fetal tracheal and rat tracheal epithelia respectively, and MNNG also induced hyperplasia and squamous metaplasia in human fetal tracheal adenoepithelium. The data suggest that 1) arsenic may be carcinogenic to the human respiratory tract but not to the rat; and 2) human tissues in organ culture are very useful for detecting carcinogens and for studying carcinogenesis.

Topics: Animals; Arsenic; Arsenic Trioxide; Arsenicals; Benzo(a)pyrene; Carcinogens; Epithelium; Fetus; Humans; Metaplasia; Methylnitronitrosoguanidine; Organ Culture Techniques; Oxides; Precancerous Conditions; Rats; Trachea; Tracheal Neoplasms

Preneoplastic transformants were isolated from primary rat tracheal epithelial cells after treatment with (a) mutagenic concentrations of the alkylating agent N-methyl-N-nitro-N'-nitrosoguanidine, (b) nonmutagenic concentrations of the DNA hypomethylating agent 5-azacytidine, or (c) after arising spontaneously. We have addressed the question of whether preneoplastic transformants induced by different carcinogens differ in their ability to progress to the immortal stage and to become neoplastic. Spontaneous transformants occurred with a very low frequency, and 5-azacytidine induced preneoplastic transformants half as efficiently as N-methyl-N-nitro-N'-nitrosoguanidine. However, no phenotypic differences could be detected between the 70 preneoplastic colonies isolated from the 3 groups; colony size, cell density, and clonogenicity were not statistically different. Clones from all 3 groups became immortal and further progressed to become neoplastic with similar frequencies. The level of expression of the oncogenes H-ras, K-ras, and raf was also similar in all 3 groups. These experiments indicated that there was no difference in the ability of spontaneous transformants or those induced by N-methyl-N-nitro-N'-nitrosoguanidine or 5-azacytidine to progress to become immortal or neoplastic. This suggests that whereas the nature of the carcinogen influenced the frequency of the initial transforming event, progression to the neoplastic stage was independent of the nature of the transforming insult.

Topics: Animals; Azacitidine; Carcinogens; Cell Transformation, Neoplastic; Epithelium; Methylnitronitrosoguanidine; Precancerous Conditions; Rats; Trachea; Tracheal Neoplasms

Following carcinogen exposure in vitro, normal rat tracheal epithelial cells are transformed in a multistage process in which the cultured cells become immortal and, ultimately, neoplastic. Five cell lines derived from tumors produced by neoplastically transformed rat tracheal epithelial cells were examined for the expression of 11 cellular oncogenes previously implicated in pulmonary or epithelial carcinogenesis. RNA homologous to fms was expressed at a level 5-19 times higher than normal tracheal epithelial cells in three of five of the tumor-derived lines. All three lines expressing high levels of fms-related RNA gave rise to invasive tumors of epithelial origin when injected into nude mice. Increased expression of the fms-related mRNA was not due to gene amplification, and no gene rearrangement was detected by Southern analyses. RNA blot analysis using a 3' v-fms probe detected a 9.5-kilobase message in the three tumor-derived lines, whereas both normal rat alveolar macrophages and the human choriocarcinoma line BeWo expressed a fms transcript of approximately 4 kilobases. We conclude from these data that the gene expressed as a 9.5-kilobase transcript in these neoplastic epithelial cells is a member of a fms-related gene family but may be distinct from the gene that encodes the macrophage colony-stimulating factor (CSF-1) receptor.

Topics: Animals; Base Sequence; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Epithelium; Gamma Rays; Gene Amplification; Genes; Methylnitronitrosoguanidine; Oncogenes; Protein-Tyrosine Kinases; Rats; Trachea; Tracheal Neoplasms

N-Ethyl-N'-nitro-N-nitrosoguanidine [(ENNG) CAS: 63885-23-4] was administered to 5 Macaca monkeys (Macaca mulatta and M. irus) at a concentration of 200 or 300 micrograms/ml for 11-26 months in their drinking water. Gastric carcinomas in the pyloric region were observed in all 5 monkeys between experimental months 11 and 38. Histologically, these carcinomas were mainly poorly differentiated adenocarcinomas and signet-ring cell carcinomas, and a few moderately and well-differentiated adenocarcinomas were also found. The macroscopic and histologic appearances of these carcinomas were similar to those in humans.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Animals; Female; Femoral Neoplasms; Macaca fascicularis; Macaca mulatta; Male; Methylnitronitrosoguanidine; Osteosarcoma; Stomach Neoplasms; Tracheal Neoplasms

The purpose of the studies described in this and the accompanying paper (D.J. Fitzgerald et al., Cancer Res., 46:4642-4649, 1986) was to define the transformed colonies of the rat tracheal epithelial cell transformation system (see P. Nettesheim and J.C. Barrett, CRC Crit. Rev. Toxicol., 12: 215-239, 1984) in terms of differentiation and growth characteristics. Exposure of low-density primary rat tracheal epithelial cell cultures to N-methyl-N'-nitro-N-nitrosoguanidine results in the development of growth-altered colonies of different sizes and morphologies, which can be readily scored 5 wk after treatment. These colonies were classified into four morphological types (I to IV) based on their light microscopic and ultrastructural features. Colonies designated as types I and II were small, had a low cell density, and were composed principally of large, pale-staining (Giemsa) flattened cells with a low nuclear/cytoplasmic ratio. Examination of the fine structure of these colonies revealed a monolayer of extremely attenuated cells containing well-developed Golgi complexes, endoplasmic reticulum, numerous secondary lysosomes, but few tonofilaments and desmosomes. Colonies of types III and IV were large, basophilic (Giemsa), high-density colonies, consisting primarily of closely packed, small, round cells with high nuclear/cytoplasmic ratio. Analysis of the fine structure of these colonies revealed two to four stratified layers of poorly differentiated cells and cells having features of keratinocyte differentiation, as evidence by abundant tonofilament bundles, well-developed desmosomes, and occasional keratohyaline granules. Differential cell counts were carried out at the ultrastructural level on pooled cells from each colony type (except type I); approximately 90% of the cells from type II colonies were large, electron-lucent cells, while this cell type comprised only 20 to 30% of the cells of colonies of types III and IV. The predominant cell type (approximately 60%) in colonies of types III and IV showed clear signs of keratinocyte differentiation. Type IV colonies contained a significantly larger proportion (greater than 20%) of small, poorly differentiated cells than type III (6%) and type II (1%) colonies. A combined autoradiographic and ultrastructural study revealed that the small, poorly differentiated cells were most active in synthesizing DNA and had a labeling index of 60%. Other cell types were far less frequently labeled, particularly the large,

Topics: Animals; Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Methylnitronitrosoguanidine; Microscopy, Electron; Microscopy, Electron, Scanning; Rats; Time Factors; Tracheal Neoplasms

The purpose of the studies described here was to define the biological behavior of the various clonally transformed colonies observed in cultures of carcinogen-exposed rat tracheal epithelial cells. As described in the preceding paper (H. Kitamura et al., Cancer Res., 46: 4631-4641, 1986), these colonies fall into four morphologically distinct categories. In the studies reported here we found that type I colonies had the smallest growth fraction (7%) and contained the lowest frequency of clonogenic cells (approximately 10(-3)). Colonies of types II to IV had mean growth fractions of 21 to 28%, and the frequency of clonogenic cells was 2 to 5 X 10(-2) when measured under growth-permissive conditions (3T3 feeders). When the clonogenic cell assays were performed under selective conditions to identify cell variants which can grow without feeder support, the average frequency of such clonogenic cells in type I colonies was less than 4 X 10(-5) and in colonies of types II to IV, between 5 X 10(-4) and 10(-2). In type IV colonies, the total number of cells per colony increased 8-fold between 5 and 12 wk postcarcinogen, but the clonogenic cell compartment increased 42-fold; the compartment of variant clonogenic cells, which are able to replicate on plastic, increased 139-fold during the same period of time. This indicated that major changes in the self-renewal capacity of the clonogenic cells were taking place during this early stage of transformation. Examination of the daughter colonies produced by replating colonies of types I to IV revealed that clonogenic cells with different growth potential existed within the same parent colony. Comparison of transformed colonies of the same type showed a marked degree of heterogeneity in the sizes of growth fractions and clonogenic cell fractions. These studies further indicated that, within all colonies, including the most advanced transformants, the majority of the cells were nonreplicating, terminal cells, suggesting that, at least during early stages of transformation, the transformed characteristics were not transferred from parent to daughter cells. With the exception of type I colonies, most of the colonies recognizable at 5 wk after carcinogen exposure progressed with time and acquired the morphological characteristics of type IV colonies, which were the most transformed phenotype. We conclude that transformation of rat tracheal epithelial cells is an asynchronous process and that the morphologically distinct type

Topics: Animals; Cell Cycle; Cell Transformation, Neoplastic; Epithelium; Methylnitronitrosoguanidine; Neoplastic Stem Cells; Rats; Time Factors; Tracheal Neoplasms

In summary, two in vitro systems with rat tracheal epithelial cells were used to demonstrate that initiated respiratory tract epithelial cells can be promoted or enhanced to transform at a higher frequency by exposure to a noncarcinogenic tumor promoter. The latter two studies suggest possible mechanisms for this enhancement: 1) TPA amplifies the chromosomal instability brought about by initiating doses of carcinogen and 2) TPA alters gene expression and cellular differentiation such that it causes the initiated cell to escape the normal differentiation and senescence process thereby enhancing the proliferative lifespan of initiated cells.

Topics: Aneuploidy; Animals; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Keratins; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Tetradecanoylphorbol Acetate; Tracheal Neoplasms

The development of transformed colonies and concomitant changes in proliferative and nonproliferative cell compartments were studied in rat tracheal epithelial (RTE) cell cultures following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Primary RTE cells were plated onto 3T3 feeder layers and treated with MNNG (0.25 micrograms/ml) or solvent. Seven days later, the feeder cells were removed to select for enhanced growth variants, which are the transformants of the RTE cell system, usually scored 5 weeks after carcinogen exposure. Most of the RTE cell colonies, which originally formed during the first 7 days of culture, disappeared within 2 weeks after feeder cell removal in control and MNNG-treated cultures. In control cultures, about 3% of the original colonies persisted, while in MNNG-treated cultures, a larger percentage (approximately 9%) of the colonies persisted. These percentages remained constant from 3 to 7 weeks. Based on colony size, cell density, and cell morphology, the persistent colonies were classified into transformed colonies (large colony size, high cell density, high nuclear:cytoplasmic ratio) and untransformed colonies (small size, low cell density, low nuclear:cytoplasmic ratio). In the MNNG-treated cultures, about 50% of all persistent colonies showed transformed morphology. Their frequency remained unchanged between 3 and 7 weeks of culture. In contrast, only 10 to 15% of the persistent colonies in control cultures showed transformed morphology at 3 weeks, but that proportion increased steadily between 3 and 7 weeks. These data suggest that, in control cultures, transformed colonies developed spontaneously as a function of time within untransformed colonies. Autoradiographic studies with [3H]thymidine showed that labeling indices in the early "normal" RTE cell colonies between Days 4 and 7 of culture were very high, ranging between 75 and 90%. In contrast, the labeling indices of persistent colonies, both those without and those with transformed morphology, were low, i.e., between 18 and 25%, indicating that a major proportion of cells was either noncycling or cycling very slowly. The relative compartment sizes of cells with stem cell characteristics and of cells with characteristics of transformed stem cells were estimated before and after transformed colonies appeared.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Male; Methylnitronitrosoguanidine; Precancerous Conditions; Rats; Rats, Inbred F344; Stem Cells; Thymidine; Trachea; Tracheal Neoplasms

Studies were carried out to examine the susceptibility of normal and initiated tracheal epithelial cells of rats to known tumour-promoting agents. The ability of normal rat tracheal epithelial cells to form colonies in cultures was enhanced markedly by addition of known tumour promoters to the medium. Several retinoids were shown to reduce the ability of these cells to form colonies in culture and to inhibit this effect of 12-O-tetradecanoylphorbol-13-acetate (TPA). A transformation assay of tracheal epithelial cells was used to study promotional effects of TPA in cultures initiated with N-methyl-N'-nitro-N-nitrosoguanidine. In this assay, four stages of transformation are recognized. TPA did not affect measurably the first two transformation stages, i.e., the development of transformed colonies (enhanced growth variants) and the 'immortalization' of enhanced growth variant-derived subcultures. However, treatment of cultures with TPA during the early post-initiation period resulted in a marked enhancement of the appearance of the third transformed phenotype, which is characterized by anchorage independence of growth. The findings of these in-vitro initiation-promotion studies paralleled, in all major respects, the results obtained in in-vivo - in-vitro studies. Tracheas exposed in vivo to initiator and promoter were shown to develop the same transformants observed in the in-vitro assay. TPA affected primarily the late anchorage-independence phenotype. Parallel tumour induction studies showed that TPA increased markedly the incidence of tracheal carcinomas following initiation with a low dose of 7,12-dimethylbenz[a]anthracene. The studies thus demonstrate that TPA is an effective tumour promoter for rat tracheal epithelium, causing an increase in tracheal carcinomas. They further suggest that the action of TPA on an early transformed cellular phenotype enhances the development of later phenotypes, including neoplastic cell variants.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Epithelium; Humans; Methylnitronitrosoguanidine; Mice; Rats; Respiratory Tract Neoplasms; Tetradecanoylphorbol Acetate; Trachea; Tracheal Neoplasms

The tracheal epithelium of the Fischer 344 rat is histologically very similar to that of the human bronchus. Also, carcinomas of tracheal origin in F-344 rats are similar in morphology to human bronchogenic carcinomas. Tumor promotion in rat tracheal epithelium was studied by using two model systems. The first is a heterotopic transplant system in which rat tracheas are implanted subcutaneously on the backs of isogenic recipents. In the first system, the epithelium was topically exposed to pellets containing 7,12-dimethylbenz(a)anthracene (DMBA), used as the initiating agent, followed by pellets containing the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the promoting agent. After 98 weeks, a three- to fourfold increase in the percentage of tracheas having malignant tumors was seen in tracheal transplants receiving both DMBA and TPA compared to DMBA alone. Exposure of the tracheal grafts to TPA alone resulted in epithelial hyperplasia and inflammation, but no dysplastic lesions. The second system is an organ culture-cell culture system in which small pieces of trachea are grown in organ culture, then epithelial cells are grown from these pieces as primary cell cultures. The organ cultures were exposed to the direct alkylating agent, N-methyl-N'-nitro-N -nitrosoguanidine (MNNG) used as the initiator, then multiple short exposures to TPA were used to promote. Primary cell cultures and cell lines were then established from these explants. After 52 weeks, a five-fold increase in the percentage of explants producing tumorigenic cell lines was observed when MNNG + TPA-exposed explants were compared to MNNG-exposed explants. Tracheal explants exposed to TPA alone produced many cell lines but none tested were tumorigenic. These two systems provide a means to study tumor promotion in respiratory epithelium. The evidence more importantly suggests that airborne promoting substances may play a key role in the development of bronchogenic carcinoma.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cells, Cultured; Cocarcinogenesis; Epithelium; Methylnitronitrosoguanidine; Neoplasms, Experimental; Organ Culture Techniques; Rats; Tetradecanoylphorbol Acetate; Trachea; Tracheal Neoplasms

To study the mechanisms of carcinogenesis, we have developed a system that uses normal cells from an environmentally and epidemiologically relevant tissue, respiratory epithelium. The induction of preneoplastic variants of epithelial cells in culture was quantitated on a per-cell basis following exposure of rat tracheal epithelial (RTE) cells in vitro to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Following treatment of normal RTE cells, large colonies of altered cells exhibiting an enhanced growth potential under selective culture conditions were observed, while normal RTE cells ceased proliferation after several cell doublings. After further growth in culture, these altered cells acquired the ability to grow in semisolid medium and to produce squamous cell carcinomas when injected into nude mice. The induction of enhanced growth variants of RTE cells by MNNG occurred with a high frequency (greater than or equal to 2.6%/colony-forming cell). In addition, a linear dose-response curve with a slope of approximately 1 was observed when the logarithm of MNNG-induced transformation frequency was plotted versus the logarithm of MNNG dose. These results are consistent with a one-hit mechanism for induction of preneoplastic variants of RTE cells by MNNG. Similar frequencies and kinetics of induction of preneoplastic variants in other culture systems using diploid cells have been observed, suggesting a common mechanism for this early step in carcinogenesis. The RTE cell system will be useful for mechanistic studies of early as well as late changes in the development of neoplasia by epithelial cells.

Topics: Animals; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Epithelium; Genetic Variation; Kinetics; Male; Methylnitronitrosoguanidine; Precancerous Conditions; Rats; Rats, Inbred F344; Tracheal Neoplasms

Primary cultures of rat tracheal epithelial cells were treated with the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to quantitatively study the early events during neoplastic transformation. Epithelial cells were dissociated from tracheas of specific-pathogen-free Fischer-344 rats and were plated on collagen-coated tissue culture dishes. To determine cytotoxicity, cells were exposed on day 1 to various concentrations of MNNG for 3 h and colony forming efficiency (CFE) was determined on day 7. MNNG at a concentration of 0.1 microgram/ml did not decrease CFE as compared to the control cultures, whereas 1 microgram/ml reduced the CEF by 75%. For transformation studies, primary cell cultures received single exposures to MNNG (0.1-0.6 microgram/ml) or multiple exposures to 0.1 microgram/ml of MNNG for 3 h between days 1 and 17. In carcinogen-exposed cultures, morphologically altered foci appeared on day 18, recognizable by high cell density. Transformation frequencies between 1 and 8% were observed depending on MNNG concentration. Cultures containing altered foci continued to grow during the third and fourth week when control cultures had ceased to proliferate and exfoliated from the dish. Over 40% of the cultures which received multiple exposures to MNNG acquired cell line status and could be subcultured greater than or equal to 20 times. None of the 30 control cultures became cell lines. Seventy per cent of MNNG-exposed cell lines showed the anchorage independent growth phenotype at passage 20 as judged by growth in agarose. Four of 10 cultures exposed either 6 or 8 times to MNNG formed invasive squamous cell carcinomas at passage 20 upon inoculation into nude mice. Based on these and previous studies, we feel that unrestricted cell replication is an early key event in carcinogen-exposed epithelial cell populations, preceding neoplastic transformation.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats; Rats, Inbred F344; Trachea; Tracheal Neoplasms

Two cell lines (2-10-1 and 8-10-2) derived by exposure to primary tracheal explants to MNNG in vitro were not tumorigenic in syngeneic F-344 rats or athymic BALB/c (nu/nu) mice at early passage, but became tumorigenic at late passage. These cell lines are therefore suited to study the expression of neoantigens during neoplastic development. Transplantation resistance to late-passage, tumorigenic cells was indicated in syngeneic rats using an immunization protocol of repeated cell inoculation and tumour ablation. Spleen cells from such animals were reactive in 20h microcytotoxicity assays against neoplastic cell lines, but unreactive to normal tracheal epithelial cells. Similarly, immune spleen cells co-cultivated in vitro for 6 days with irradiated neoplastic cell lines before assay for microcytotoxicity were strongly reactive, whereas co-cultivation with normal epithelial cells did not stimulate reactivity. Antibody to these neoplastic cell lines was demonstrated in sera of tumour-resistant rats by an indirect radiolabelled-antibody binding test and by indirect immunofluorescence. There was no significant binding to normal tracheal epithelial cell outgrowths.

Topics: Animals; Antibodies, Neoplasm; Cell Line; Cell Transformation, Neoplastic; Epithelium; Female; Immunity, Cellular; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Rats, Inbred F344; Tracheal Neoplasms; Transplantation Immunology; Transplantation, Isogeneic

The enhancement of chemical carcinogenesis was demonstrated in vitro in rat tracheal epithelium exposed in organ culture to N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) followed by multiple exposures to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The explants were exposed to 0 or 0.0001 mu g MNNG per ml for 6 h on days 3 and 6 of culture, and then to 0 or 1.0 mu g TPA per ml for I h on days 9, 15, 21, and 27 of culture. Primary cell cultures were then established from epithelial outgrowths of these explants. Morphologically altered cells were seen in the primary cell cultures derived from MNNG + TPA-exposed explants on an average of 130 days after MNNG exposure. In primary cultures derived from explants exposed to either TPA or MNNG alone, morphologically altered cells were seen at 190 days and 210 days, respectively. No morphologically altered cells were seen in solvent control primary cell cultures. Only cultures containing morphologically altered cells could be repeatedly subcultured. Cell lines established from these cultures were inoculated i.m. into immunosuppressed isogeneic hosts at 150, 200, 250, 310, and 365 days following carcinogen exposure. All five explants exposed to both MNNG and TPA produced at least one cell line which formed carcinomas when inoculated 365 days following exposure, while only two of the five explants produced tumorigenic cell lines in the MNNG-only group. Cell lines from explants exposed to both MNNG and TPA were tumorigenic an average of 90 days earlier than cell lines from explants exposed to MNNG alone. None of the cell lines obtained from the explants exposed only to TPA were tumorigenic. These studies show that, following carcinogen exposure, TPA can enhance the oncogenesis of cultured respiratory tract epithelium in terms of increasing the frequency of transformation and decreasing the time at which transformation could first be observed.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Female; Methylnitronitrosoguanidine; Neoplasms, Experimental; Phorbols; Rats; Tetradecanoylphorbol Acetate; Time Factors; Trachea; Tracheal Neoplasms

Topics: Adenocarcinoma; Animals; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Epithelium; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats; Time Factors; Tracheal Neoplasms