Metastasis from squamous cell carcinomas of SENCAR mouse skin produced by complete carcinogenesis

The incidence of metastasis was evaluated in female SENCAR mice after induction of squamous cell carcinomas by repetitive applications of either benzo [a] pyrene (B [a] P) or N-methyl-N'-nitro-N-nitrosogaunidine (MNNG). Between 41 and 50 weeks 50% of the animals with carcinomas in the B [a] P group had metastases, whereas 20% had metastases in the MNNG group. Very few metastases were observed before 40 weeks of treatment. The major site of metastasis was the lungs; however, metastatic tumors were also found in lymph nodes, adrenal glands and kidneys.     

Expression of cyclooxygenase-1 and cyclooxygenase-2 in bronchial epithelium and nonsmall cell lung carcinoma

BACKGROUND: Cyclooxygenase (Cox) is the main target enzyme for the nonsteroidal antiinflammatory drugs that have been shown to suppress carcinogenesis in both experimental models and epidemiologic studies. METHODS: To evaluate its utility as an intermediate biomarker in bronchial chemoprevention trials, the authors examined Cox 1 and Cox 2 expression in normal and premalignant bronchial epithelial cells and nonsmall cell lung carcinoma (NSCLC) samples using an immunohistochemical staining technique. Included in the current study were 101 NSCLC samples and 77 bronchial biopsy samples obtained from 15 healthy smokers. RESULTS: In the normal bronchial epithelium, Cox 2 expression was found to be completely negative whereas Cox 1 expression was noted in a few scattered cells. The areas of basal cell hyperplasia and squamous metaplasia demonstrated the same pattern. There were relatively more Cox 2-positive tumors, as defined by positive staining in > 10% of tumor cells, than Cox 1-positive tumors (30 of 101 tumors [30%] vs. 14 of 101 tumors [14%]; P = 0.01). When tumor types were considered, there were more Cox 2-positive adenocarcinomas compared with squamous cell carcinomas (21 of 51 adenocarcinomas [41%] vs. 9 of 46 squamous cell carcinomas [20%]; P = 0.03). In contrast, fewer adenocarcinomas tended to show Cox 1 expression compared with squamous cell carcinomas (4 of 51 adenocarcinomas [8%] vs. 9 of 46 squamous cell carcinomas [20%]; P = 0.14). Although smokers tended to have more Cox 2-positive tumors than nonsmokers (29 of 91 tumors in the smokers [32%] vs. 1 of 10 tumors in the nonsmokers [10%]; P = 0.15), there was no statistically significant relation found between Cox 1 or Cox 2 expression and smoking status or prognostically significant clinicopathologic features. CONCLUSIONS: The results of the current study suggest that Cox 1 and Cox 2 expression may not be a useful intermediate biomarker in bronchial chemoprevention trials. Nevertheless, considering the patterns of Cox 1 and Cox 2 expression in tumor cells, Cox expression status may be a useful parameter when designing treatment strategies for a subset of NSCLC patients.     

Identification and cloning of two overexpressed genes, U21B31/PRAD1 and EMS1, within the amplified chromosome 11q13 region in human carcinomas.

Amplification of the chromosome 11q13 region is frequently found in human breast cancer and in squamous cell carcinomas of the head and neck, and has been associated with an unfavourable clinical course of disease. The known oncogenes within the amplified 11q13 region, INT2 and HSTF1, are rarely expressed in these tumours, indicating that another, hitherto unidentified, gene or genes confer(s) the biological (prognostic) significance to the amplification of the 11q13 region. To identify the gene or genes, we have constructed a cDNA library from a cell line with an 11q13 amplification and have performed differential cDNA cloning using [32P]dCTP-labelled cDNAs from human squamous cell carcinoma cell lines with and without an 11q13 amplification. We isolated two cDNA clones, U21B31 and U21C8, which recognize two genes amplified and overexpressed in cell lines harbouring an 11q13 amplification. In breast carcinomas and in squamous cell carcinomas amplification of both the U21B31 and the U21C8 gene was found in most tumours with an amplification of the 11q13 region, despite the large distance between both genes. Sequence analysis of the U21C8 cDNA clone revealed no homology to known genes; we call this gene EMS1. The U21B31 cDNA clone corresponded to the 3' end of the PRAD1 proto-oncogene, recently cloned from a parathyroid adenoma. Both gene products are of interest as potential markers to identify tumours with an 11q13 amplification.     

Murine squamous cell carcinoma cell lines produced by a complete carcinogenesis protocol with benzo[a]pyrene exhibit characteristic p53 mutations and the absence of H-ras and cyl 1/cyclin D1 abnormalities

In this report, we describe the isolation and characterizationof six murine squamous cell carcinoma cell lines (BPCC) derivedfrom carcinomas produced by a complete carcinogenesis protocolwith benzo[a]pyrene (B[a]P). All six cell lines were tumorigenicto varying degrees in nude mice, and several were spontaneouslymetastatic to the lungs. The in vivo invasive potential of eachBPCC cell line was determined using de-epithelialized trachealxenotransplants into which cells were inoculated. This assayrevealed positive association of tumor grade with in vivo invasiveness,yet no clear relationship to the spontaneous metastatic potentialof the cell lines, suggestive that invasive potential is onlyone determinant of the overall metastatic phenotype. At themolecular level, all six BPCC cell lines revealed the absenceof mutations in the H-ras oncogene and no amplification or rearrangementin the cyl l/cyclin Dl putative oncogene. Analysis of the p53tumor suppressor gene revealed a direct correlation betweenpositive nuclear immunohistochemical staining of the p53 proteinin four BPCC cell lines and the presence ofp53 mutations identifiedby direct sequence analysis. The localization of mutations toexons 7 and 8 of the p53 gene and the detection of G to T transversionsin two of the four cell lines bearing p53 mutations are in agreementwith previous analyses of a large series of primary B[a]P-inducedmurine skin tumors. In addition, frameshift mutations were identifiedin two cell lines. The correlation of the biological and molecularproperties of these BPCC cell lines with the known characteristicsof primary squamous cell carcinomas induced by B[a]P indicatesthat these cell lines could be useful tools in elucidating themechanisms of tumorigenesis of this important chemical carcinogen.     

Mutagenesis and carcinogenesis induced by dibenzo[a,l]pyrene in the mouse oral cavity: a potential new model for oral cancer

Cancer of the oral cavity is a serious disease, affecting about 30,000 individuals in US annually. There are several animal models of oral cancer, but each has certain disadvantages. As a new model, we investigated whether topical application of the tobacco smoke carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) is mutagenic and carcinogenic in the oral cavity of the B6C3F1 lacI and B6C3F1 mouse, respectively. B6C3F1 lacI mice received DB[a,l]P (0, 3, 6, 12 nmol) 3× per week. B6C3F1 mice received the same doses and also 24 nmol. At 38 weeks mutagenesis was measured in oral tissues in lacI mice. For the high dose group, the mutant fraction (MF) in upper mucosa and tongue increased about twofold relative to that in vehicle-alone. The increases were statistically significant. The mutational profile in the DB[a,l]P-induced mutants was compared with that induced by benzo[a]pyrene (BaP) in oral tissue. BaP is mutagenic in many tissues when administered by gavage. The mutational profile for DB[a,l]P was more similar to that reported for p53 mutations in head and neck cancers than was that of BaP. At 47 weeks, oral squamous cell carcinomas (OSCC) were found in 31% of the high-dose B6C3F1 group. Elevations of p53 and COX-2 protein were observed in tumor and dysplastic tissue. As DB[a,l]P induces mutations and tumors in the oral cavity, and has a mutational profile in oral tissue similar to that found in p53 in human OSCC, the treatment protocol described here may represent a new and relevant model for cancer of the oral cavity.     

Detection of squamous cell carcinoma antigen in normal squamous epithelia and in squamous cell carcinomas of the uterine cervix

Squamous cell carcinoma (SCC) antigen is a subfraction of tumor antigen TA-4 isolated from a cervical squamous cell carcinoma. The specificity of SCC antigen and the factors influencing its release into serum were evaluated. Antigen concentrations were measured in 157 tissue extracts and in 188 sera of patients with nonmalignant or malignant gynecologic diseases. A commercial radioimmunoassay based on polyclonal antibodies (Abbott Laboratories, North Chicago) was used. Cytosol concentrations were significantly higher (P less than 0.005) in normal squamous epithelia (means = 6040 ng/mg cell protein [CP]) and in squamous cell carcinomas (means = 2483 ng/mg CP) of the exocervix than those in normal columnar epithelia and in adenocarcinomas of the endocervix, endometrium, ovary, and breast (means = 1-508 ng/mg CP). Despite the high antigen concentrations in normal squamous epithelia, elevated serum levels (greater than 2.5 ng/ml) were almost exclusively found in patients with cervical squamous cell carcinomas. The sensitivity of SCC antigen as a marker for primary carcinomas was 61%, increasing from 29% in Stage I to 89% in Stage IV. The positivity rate was higher in women with well-differentiated (78%) and moderately differentiated carcinomas (67%) than in those with poorly differentiated tumors (38%). The results show that SCC antigen is not tumor specific. The release into serum is independent of local tissue content, but is apparently influenced by the infiltrative growth, the mass, and the degree of histologic differentiation of the tumor.     

Inhibition of DNA synthesis and neoplastic cell growth by vitamin A (retinol)

The inhibitory effect of vitamin A alcohol (retinol) on the DNA synthesis and neoplastic cell growth of chemically induced carcinomas (squamous cell carcinomas in Swiss male albino mice and basal cell carcinomas in inbred SD rats) by 3-methylcholanthrene [(MCA) CAS: 56-49-5] was studied. A marked inhibition of squamous cell carcinomas and basal cell carcinomas was observed following a combined administration of MCA and vitamin A (retinol) compared to the finding for animals treated with MCA alone (P less than .001). DNA radioactivity and autoradiographic studies with the use of [3H]thymidine showed a marked inhibition of DNA synthesis (twofold to threefold) in the neoplastic cell nuclei following a concomitant administration of vitamin A (retinol) and MCA (12%) as compared to the DNA synthesis following administration of MCA alone (31%) (P less than .001). Electron microscopic and cytologic observations revealed an advanced cytolysis and disorganization of neoplastic cells with reduction of polysomes, tonofilaments, and lysosome populations and mitochondrial alterations following vitamin A and MCA administration as compared to characteristic squamous neoplastic cells and basal neoplastic cells following MCA treatment alone. In addition, scanning electron microscopy revealed advanced changes of cell surfaces with reduction of microvilli and disorganization of cytoarchitecture. The present findings demonstrate that vitamin A exerts its anticarcinogenic effect by inhibiting DNA synthesis, disrupting cell surfaces, and possibly interfering with MCA metabolism in epidermal cells.     

The EP1 receptor for prostaglandin E2 promotes the development and progression of malignant murine skin tumors

High levels of prostaglandin E2 (PGE2) synthesis resulting from the up-regulation of cyclooxygenase (COX)-2 has been shown to be critical for the development of non-melanoma skin tumors. This effect of PGE2 is likely mediated by one or more of its 4 G-protein coupled membrane receptors, EP1-4. A previous study showed that BK5.EP1 transgenic mice produced more carcinomas than wild type (WT) mice using initiation/promotion protocols, although the tumor response was dependent on the type of tumor promoter used. In this study, a single topical application of either 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P), alone, was found to elicit squamous cell carcinomas (SCCs) in the BK5.EP1 transgenic mice, but not in WT mice. While the epidermis of both WT and transgenic mice was hyperplastic several days after DMBA, this effect regressed in the WT mice while proliferation continued in the transgenic mice. Several parameters associated with carcinogen initiation were measured and were found to be similar between genotypes, including CYP1B1 and aromatase expression, B[a]P adduct formation, Ras activity, and keratinocyte stem cell numbers. However, EP1 transgene expression elevated COX-2 levels in the epidermis and SCC could be completely prevented in DMBA-treated BK5.EP1 mice either by feeding the selective COX-2 inhibitor celecoxib in their diet or by crossing them onto a COX-2 null background. These data suggest that the tumor promoting/progressing effects of EP1 require the PGE2 synthesized by COX-2.     

The effects of weak or non-carcinogenic polycyclic hydrocarbons on 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene skin tumor-initiation.

Benzo[e]pyrene (B[e]P) inhibited 7,12-dimethylbenz[a]anthracene (DMBA) skin tumor-initiation in mice by 84%, whereas pyrene and fluoranthene inhibited DMBA initiation by 50 and 34%, respectively. However, B[e]P, pyrene and fluoranthene had either no significant effect or a slight enhancing effect on benzo[a]pyrene (B[a]P) skin tumor-initiation. In addition, B[e]P had essentially no effect on the initiating ability of (+/-)B[a]P-7 beta,8 alpha-diol-9 alpha,10 alpha-epoxide. As a tumor-initiator, B[e]P was found to have very weak activity at a 252 microgram/level (0.4 papillomas/mouse at 40 weeks) and no activity at 100 microgram. When given at a dose of 100 microgram twice weekly, B[e]P induced 2.1 papillomas/mouse at 30 weeks, and 25% of the mice had carcinomas at 40 weeks. However, B[e]P carcinogenic activity is weak when compared to B[a]P, which can induce a comparable tumor response at a dose of 5 microgram twice weekly. When B[e]P was tested as a tumor promoter at a dose of 100 microgram twice weekly after DMBA initiation, it induced 4.5 papillomas/mouse at 30 weeks and a 45% carcinoma incidence at 40 weeks, which was approximately twice as effective as B[e]P alone. The data show that B[e]P is a very weak tumor initiator, a weak complete carcinogen, a moderate tumor promoter, possibly a weak co-tumor-initiator when given with B[a]P, and a potent anit-tumor-initiator when given with DMBA. The anti-tumor initiating and co-tumor-initiating effects of B[e]P appear to be related to its ability to modify the conversion of the tumor initiator into an electrophilic intermediate(s) which are capable of covalently binding to DNA. In addition, B[e]P induced epidermal cellular proliferation which may be related to its promoting ability.     

Induction of squamous cell carcinoma in the rat lung by 1,6-dinitropyrene

The carcinogenicity of 1-nitropyrene [(1-NP) CAS: 5522-43-0] and 1,6-dinitropyrene [(1,6-DNP) CAS: 42397-64-8] was examined by their direct injection in a beeswax-tricaprylin vehicle into the lung of male F344/DuCrj rats. Of 28 rats given 0.15 mg of 1,6-DNP, 21 (75%) developed squamous cell carcinomas, 2 (7%) developed undifferentiated carcinomas, and 2 (7%) had squamous metaplasias in the lung by 72 weeks. In 32 rats that received 1.5 mg of 1-NP, neither carcinoma nor squamous metaplasia was induced. In all 19 rats (100%) given 0.5 mg of 3-methylcholanthrene [(MCA) CAS: 56-49-5], squamous cell carcinomas were induced earlier than in rats treated with 1,6-DNP. In 1 of 31 rats (3%) given the beeswax-tricaprylin vehicle only, squamous metaplasia was induced. Distant metastases of induced tumors were observed in 4 rats treated with 1,6-DNP and in 1 rat receiving MCA. Two lung tumors induced by 1,6-DNP were successively transplanted into the same strain of rats for 3 generations.     

Promotion pattern and tumour types in two-stage rat skin carcinogenesis.

Carcinogenic studies were performed on the skin of inbred male Lister rats using 7,12-dimethylbenz[alpha]anthracene (DMBA) in low doses (2.5 mg and 5.0 mg), followed by promotion with croton oil (CO) in one half of the animals. The clinically observed tumour rate did not differ after the two carcinogen doses and was practically unaffected by promotion, only a marginal increase in effect being noted after DMBA 2.5 mg. Also the latency periods were alike. The total tumour crop was increased by promotion, but histologically the predominant clinical skin tumour was a sebaceous proliferation with or without associated papillomatous squamous cell hyperplasia. This lesion did not in microscopic structure or cellular characteristics fulfil the criteria of a neoplastic tumour. Basal cell carcinomas, adnexal tumours and pure squamous cell tumours were rare and were not promoted by CO. CO had a marked promotion effect, but only on the mixed sebaceous-papillomatous hyperplasias, which were more than doubled in number. In conclusion, carcinogenesis was weak after the low DMBA-doses, and promotion by CO of neoplastic tumours was not seen.     

Benzo[a]pyrene--DNA adduct formation in target cells in a cell-mediated mutation assay

In order to analyze the adducts formed in V79 Chinese hamstertarget cells in a Syrian hamster embryo (HE) cell-mediated mutationassay, a procedure was developed in which the HE cells are differentiallykilled when the mixed cell suspension is treated with antiserumto Syrian HE cells and complement. The V79 cell suspension,separated from lysed HE cells by gradient centrifugation, is> 90% HE cell-free. When the carcinogen - DNA interactionproducts formed in these two cell types were analyzed afterexposure to [³H]benzo[a]pyrene (B[a]P) for 24 h under the conditionsof a cell-mediated mutation assay, the major B[a]P-DNA adductsin both cell types resulted from reaction of the anti and synisomers of B[a]P-7,8-diol-9,10-epoxide with DNA. The amountof B[a]P bound/mg DNA in the target cells was only 30% lessthan in the activator cells, and the relative proportions ofthe adducts of the syn and anti isomers were similar in thetwo cell types. The target cells, which do not metabolize B[a]P,were also unable to metabolically activate B[a]P-7,8-diol toDNA-binding metabolites. Thus, the transfer of activated B[a]Pmetabolites from activator cells to the DNA of target cellsis a relatively efficient process that appears to be independentof the relative reactivity of specific metabolites with cellularnucleophiles. The immunological cell separation procedure wedescribe can be adapted to the analysis of carcinogenl-cellinteraction products formed in cell-mediated assays in whichother types of activator and target cells are used to measureeither mutation or another biological endpoint.     

A comparison of the tumors induced by coal tar and benzo[a]pyrene in a 2-year bioassay

The tumorigenicity of two coal tar mixtures was compared to that of benzo[a]pyrene after 2 years of feeding. Mixture 1, a composite of coal tar from seven coal gasification plant waste sites, was fed to female B6C3F1 mice (48 mice per group) for 2 years at doses of 0.0, 0.01, 0.03, 0.1, 0.3, 0.6 and 1.0%. Mixture 2, which was composed of coal tar from two of the seven waste sites and another site having a high benzo[a]pyrene content, was fed at doses of 0.0, 0.03, 0.1 and 0.3%. Additional groups of mice were fed 0, 5, 25 and 100 ppm benzo[a]pyrene. The coal tar diets induced a dose-related increase in hepatocellular adenomas and carcinomas, alveolar/bronchiolar adenomas and carcinomas, forestomach squamous epithelial papillomas and carcinomas, small intestine adenocarcinomas, histiocytic sarcomas, hemangiosarcomas in multiple organs and sarcomas. Benzo[a]pyrene treatment resulted in an increased incidence of papillomas and/or carcinomas of the forestomach, esophagus and tongue. A comparison of the results indicated that the benzo[a]pyrene in the coal tar diets could be responsible for the forestomach tumors. In contrast, the lung and liver tumors appeared to be due to other genotoxic components contained within the coal tar mixture, while the small intestine tumors resulted from chemically- induced cell proliferation that occurred at high doses of coal tar.      

Cell proliferation and histologic classification of bronchogenic carcinoma.

The rate of cell proliferation of 99 bronchogenic carcinomas (94 primary tumors and 5 metastases) was evaluated from the labeling index after in vitro incorporation of [3H]thymidine; the rate was then correlated with the histologic tumor type according to the classification of the World Health Organization (WHO). Cell proliferation was significantly slower in adenocarcinoma (WHO type III) than in squamous cell carcinoma (WHO type I), small cell anaplastic carcinoma (WHO type II), and large cell carcinoma (WHO type IV). Cells proliferated at a significantly higher rate in large cell carcinoma than in the squamous cell type, whereas no significant difference was observed between the other cell types. Dedifferentiated forms of squamous cell carcinomas had a higher rate of cell proliferation than did differentiated forms of the same cell type. Metastases of small cell anaplastic carcinoma did not differ in cell proliferation from primary tumors of the same cell type.     

Serum haptoglobin levels in mice with 7, 12-dimethylbenz[a]anthracene-induced tumors.

Serum haptoglobin (Hp) levels were determined periodically in mice treated with the carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA). The magnitude and duration of the Hp response to tumors induced by DMBA were dependent on the tumor type; lymphocytic lymphomas elicted a minimal response, whereas mice bearing mammary carcinomas and stomach squamous cell carcinomas had high Hp levels which remained elevated throughout most of the period of tumor development. In the majority of mice with mammary carcinomas, the initial rise in serum Hp coincided fairly closely with the first appearance of palpable masses. Some variation in the magnitude of the Hp response was observed between individual mice bearing chemically-induced tumors of similar histological types.     

Effect of prolonged insulin treatment on carcinoma formation in mice

Long-term insulin administration enhanced the incidence and development of chemically induced [3-methylcholanthrene (3-MCA)] squamous cell carcinomas in Swiss male mice. DNA radioactivity and light and electron microscopic autoradiography revealed that insulin administration significantly increased (2-fold) the [3H]thymidine incorporation and intracellular distribution in the neoplastic nuclei (38.50%) as compared to that of neoplastic nuclei treated with MCA alone (20%). Electron microscopic autoradiography showed a heavy [3H]thymidine distribution as developed grains over dense chromatin (heterochromatin) of neoplastic cell nuclei. Ultrastructural and cytological studies of insulin-treated carcinomas revealed the predominance of less differentiated squamous cell carcinomas with numerous vacuoles, polysomes, laminated concentric myelin figures, and phagolysosomes as compared to well differentiated squamous neoplastic cells treated with MCA only. Scanning electron microscopic observations revealed in the insulin + MCA-treated tumors the predominance of rounded cells covered with several elongated microvilli and blebs as compared to polygonal cells covered with sparse and thin microvilli, horny pearls with concentric keratinized layers after MCA treatment. An intense stromal tumor reaction can be also seen following insulin and MCA treatment. These findings demonstrate that insulin in pharmacological doses stimulates the carcinoma formation, increases DNA synthesis, and affects the squamous neoplastic cell differentiation.     

High concentration of beta-defensin-2 in oral squamous cell carcinoma

We have previously reported the presence of human beta-defensin-2 (HBD-2), a peptide with antimicrobial and cytotoxic properties, in oral squamous cell carcinomas. The aim of the present study was to measure the concentration of HBD-2 in abnormal tissues such as oral squamous cell carcinomas. HBD-2 was extracted from tissue samples in the presence of retinoic acid and subjected to reversed-phase HPLC. The fraction representing peak 17 (P17) was obtained by elution using a linear gradient of acetonitrile. Amino acid sequencing and homology studies were subsequently performed, and the molecular weight of P17 was calculated to be 432702 daltons. This molecular weight was consistent with HBD-2. The concentration of HBD-2 in the oral squamous cell carcinoma samples was 3.85+/-1.87 microg/mg which was much higher than in normal oral epithelium (0.04+/-0.02 microg/mg).     

Carcinogenesis, benzo[a]pyrene metabolism, and sister chromatid exchanges in lungs of rats after intratracheal 3-methylcholanthrene

Induction of squamous cell carcinomas in F344 rats by the intratracheal instillation of carrier-free 3-methylcholanthrene [(MCA) CAS: 56-49-5] was dependent on the total dose and on the size of the crystals. With the use of MCA particles of about 1 micron in mean diameter in doses up to 25 mg, delivered as 5 doses of 5 mg each at 2-week intervals, no tumors were produced. With particles in the range of 10-300 microns, all rats receiving 25 mg developed squamous cell carcinoma of the lung. At lower doses, the tumor incidence was dose-dependent. Metabolism of [3H]benzo[a]pyrene [CAS: 50-32-8] or [3H]MCA by microsomes from lungs and livers of treated rats was increased over that of controls and remained elevated for more than 6 weeks in the lung and 2 weeks in the liver. Repeated treatment of rats did not increase the levels of enzyme activity beyond that seen after a single treatment, nor did the increased activity persist longer than was seen after a single treatment. Sister chromatid exchanges (SCE) were measured in primary cultures of lung cells and in peripheral and spleen lymphocytes from treated rats. Elevated frequencies of SCE were found in lung cells up to 6 weeks following a single treatment with MCA. Repeated treatment did not increase the frequency or increase the persistence of the SCE. No increase in exchange frequency was found in lymphocytes of any treated rats.     

Transformation of rat tracheal epithelial cells by benzo[a]pyrene and its metabolites

Benzo[a]pyrene (B[a]P) and its 7,8-dihydrodiol form were metabolically activated to malignantly transform a non-tumorigenic rat tracheal epithelial cell line in culture. The proximal carcinogenic metabolite of B[a]P, diolepoxide I, was more efficient in transforming the epithelial cell line than the intermediate or parent compound, even at a 50- or 100-fold lower concentration, respectively. Inoculations of the transformed cell lines into immunosuppressed isogenic recipients produced differentiated carcinomas similar to those which occur in humans.