Verification in syngeneic hamsters of in vitro transformation of hamster oral mucosa by 7,12-dimethylbenz(a)anthracene.
The carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) has been used to induce oral carcinogenesis of the hamster buccal mucosa in an experimental model that exhibits many of the genetic, biochemical and pathological features of human oral squamous cell carcinoma. To complement this in vivo process we have established an in vitro transformation procedure that involved the treatment of normal hamster oral mucosal keratinocytes (NHKs) with DMBA. Uptake of DMBA in NHKs was verified by observing autofluorescence of DMBA in the oral mucosal cells. Treatment doses ranged from 5, 50 and 200 ng and the NHKs were generally treated with DMBA for 1-14 days. The 200 ng dose proved to be toxic to these cells. The 5 and 50 ng treatments were found to maintain the viability of the NHKs and demonstrate anchorage-independent agarose growth, producing 18 and 40 colonies, respectively, after 14 days of treatment. Characterisation assays included determinations for cellular growth through plating efficiency, counting of cell colony number, and 3H-thymidine incorporation. Differentiation was ascertained by counting cornified cells, specification of either high or low molecular weight keratins, the percentage of cells expressing gamma glutamyl-transpeptidase (GGT), the level of p53 expression, and a determination of cell cycle. After 24 h the plating efficiency of the NHKs was found to be slightly increased following treatment with a 5 or 50 ng dose of DMBA compared to the untreated NHKs. After 14 days of incubation these doses also enhanced the number of colonies formed by the NHKs (e.g. plating efficiency). In contrast, the number of cornified cells was reduced in these colonies, while immunohistochemistry disclosed an increase in the number of NHKs expressing low molecular weight keratins, significantly lower levels of high molecular weight keratins and high levels for GGT. Flow cytometric analysis verified an increase in p53 expression (e.g. p53 wild type, 19% and p53 mutant, 66%). Cell cycle analysis of NHKs treated with DMBA (5 ng) demonstrated a shift in the number of cells in S phase (17.2%) and G2 + mitosis (11.0%). Cells from this DMBA treatment group were injected into syngeneic hamster recipient buccal pouches (10 x 10(6) cells/0.25 ml). Squamous carcinomas grew in four of six hamster buccal pouches as determined by histopathological analysis. The in vitro assay system will enhance our ability to define the genetic and molecular changes related to chemical carcinogenesis and oral malignant transformation.
Schwartz
J L
JL
Department of Oral and Maxillofacial Pathology, Howard University, College of Dentistry, Washington, DC 20059, USA.
Shklar
G
G
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D002273:Carcinogens / Q000493:pharmacokinetics / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects* / Q000473:pathology; D002610:Cheek; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D015603:Keratinocytes / Q000187:drug effects; D007633:Keratins / Q000378:metabolism; D008647:Mesocricetus; D009061:Mouth Mucosa / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009062:Mouth Neoplasms / Q000473:pathology; D009368:Neoplasm Transplantation
D002273:Carcinogens; D015127:9,10-Dimethyl-1,2-benzanthracene; D007633:Keratins