School of Dentistry

granular cell layers . Although more than 70 dif-Human papillomavirus (HPV) has been implicated ferent types are now known , HPV shows a strong in the etiology of oral and cervical cancers. Normal type specificity for anatomical location and also for oral epithelial cells at passage two were infected characteristic type of lesions. Twenty-two types are with recombinant retrovirus containing the E6/E7 commonly found to infect the human anogenital tract open reading frames of HPV type 16. The G418-se- , and these virus types appear to have a role in lected cells that were immortalized and express HPV carcinogenesis of the anogenital region, in that more 16 E6/E7 have been in culture for over 4 years and than 90% of cervical cancers have been reported to 350 passages. In contrast, the normal oral epithelial contain HPV DNA [1]. HPV types 6 and 11 are found cells did not survive the culture environment beyond in benign condylomas, while types 16, 18, 31, and 32 7 to 9 passages. Fifteen clones were selected from are frequently associated with malignant progresthe pooled population. By Northern blot analysis all sion and cervical cancer [4 -7].

The myofibroblast is found in normal tissue as well as in a wide variety of pathological processes. We have cultured myofibroblasts and dermal fibroblasts and have found that they secrete similar type-specific procollagens into the culture media. These were primarily type I and III procollagens with a predominance of type I procollagen. These patterns are distinctly different from those of smooth muscle cells, which synthesize predominantly type III procollagen. Cultured fixed cells were also examined by immunohistochemistry. Both myofibroblasts and fibroblasts stained positively with antibodies to type I and III procollagens. Reaction to type V procollagen antibodies was prominent only in the myofibroblast, as was immunostaining with anti-muscle actin antibodies. Immunostaining with desmin antibodies was negative in both cell types. By electron microscopy, the myofibroblast had well-developed dense microfilament fibers of 40–80° that were prominent in the long axes of the cells near the cellular margins. Although the myofibroblast has properties of both smooth muscle cells and fibroblasts, it appears to be most likely a modified fibroblast that has undergone differentiation, probably in response to specific signals from the extracellular matrix.

HPV infections have been previously observed in oral cancers, and inactivation of the pS3 gene has been shown to be one of the most common genetic alterations in human tumors. We examined 179 oral specimens from 70 individuals with histologic findings of either normal mucosa (n = 6) or oral disease that ranged from mild dysplasia to invasive squamous-cell carcinoma (n = 64) to determine the occurrence of both HPV infection and p53 mutations and their relationship with several clinical factors. HPV infection was detected by PCR amplification of viral DNA, and the presence of p53 mutations was assayed using the single-strand conformation polymorphism (SSCP)-PCR technique. HPV infection was found in 3 I Yo of individuals with oral disease and was not seen in healthy individuals. Mutations in exons 5, 6, 7 or 8 of the p53 gene were detected in 37.5% of patients with oral lesions and in a biopsy from I healthy individual who was a heavy smoker. Approximately one-third of lesions classified as pre-malignant (dysplasia and carcinoma in situ) and 42% of invasive carcinomas contained p53 mutations. The majority of these mutations were G:T transversions located within exons 7 and 8. Tumor tissues from 6 patients with oral lesions were found both to be HPV-16-positive and to contain p53 mutations; of these, 4 were poorly differentiated carcinomas that were diagnosed as late-stage disease. In this study, p53 mutations were detected in the early stages of cancer development. 8 I996 WileyLiss, Inc.

Myofibroblasts are cells that have features of both smooth muscle cells and fibroblasts. Myofibroblasts from rat granulation tissue were studied using a coordinated biochemical and morphological approach. These myofibroblasts were maintained in culture for more than 17 passages. After establishing that these cells were indeed myofibroblasts by immunohistochemical and ultrastructural criteria, their biochemical parameters and phenotypic stability were studied. Rat dermal fibroblasts grown under similar conditions served as controls. Biochemically, it was found that both cells produced similar types of procollagens, namely collagens I and III, in similar proportions at or near a 1:1 ratio. However, myofibroblasts at early passage produced threefold more procollagens than did fibroblasts at the same stage of passage, while similar quantities were produced in each at late passage. These observations for procollagens were confirmed by separate studies for collagens, as measured by hydroxyproline determinations. Such differences were also reflected ultrastructurally. Secretory vesicles were identified in myofibroblasts at early stage, but not at late stage of passaging. No such vesicles were seen in fibroblasts at any stage of passage. In conclusion, myofibroblasts are active secretory variants of fibroblasts which have an unstable phenotype in culture and become indistinguishable from fibroblasts at late passage.

Human papilloma virus (HPV) type 16 has an established association with anogenital carcinoma, and to some extent with human oral squamous cell carcinoma. We hypothesize that HPV type 16 is capable of inducing chromosomal and cell cycle changes in cultured oral epithelial cells. Normal human oral epithelial cells were immortalized with recombinant retrovirus containing the E6/E7 open reading frames of HPV type 16. These cells have been in culture for more than 350 passages and over 4 years. Flow cytometry demonstrated an average of 42% nuclear aneuploidy in HPV 16-immortalized cells; 16% in normal controls (probably tetrasomy). Cytogenetic analysis demonstrated significant progression of chromosomal abnormalities. Cells at early passage (plO) showed trisomy 20, with no other major changes. At passage 18, trisomy lq and monosomy 13 were seen in addition to trisomy 20. At passage 61 there were two distinct cell populations ('a' and 'b'), with multiple chromosomal changes including trisomy 5q,14,20 in one line and 7p,9q,llq in the other. Both populations had monosomy 3p, with monosomy 8p in one population and monosomy 13 in the other. At passage 136, the cells were essentially identical to population 'b' of passage 61. At this passage, mutation of the p53 gene was detected at codon 273 of exon 8, with G to T conversion (Arg to Leu). This was absent in the normal cells from which this line was developed. Passage 262 contained the two major cell populations, each with a sub-group with additional chromosomal changes such as lOp monosomy. Cells from passages 217 and 305 were injected into nude mice a year apart Both failed to produce tumors, as did normal cells. In conclusion, we present an HPV type 16-immortalized oral epithelial cell line (IHGK) with extensive and progressive chromosomal abnormalities, invasive growth in culture and yet no tumor formation in nude mice. We suggest that the question as to whether HPV alone can induce transformation is still open.

Polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of inflammatory gingivitis and periodontitis. To further study the role of PMNs in mediating gingival injury, we cocultured these cells in vitro with monolayers of human gingival epithelial cells. Scanning electron microscopy revealed that the epithelial cells were homogeneous and SDS-PAGE/immunoblot analysis identified the presence of keratins K3, K13 and the K.6/16 pair which authenticated the oral origin of the cells. Injury to the gingival cells was determined by scanning electron microscopy and measurement of cell detachment and cytolysis. Unstimulated PMNs produced minimal lysis or detachment, but PMNs stimulated by phorbol myristate acetate produced marked epithelial cell detachment without lysis, which was time- and PMN-dose-dependent. Supernatants of activated PMNs were similarly effective, indicating that the mediator was a stable soluble substance. Elastase and cathepsin G, two neutral proteases of PMN origin, produced time- and concentration-dependent detachment of gingival epithelial cells, suggesting that these enzymes may mediate this form of injury. In other studies, gingival epithelial cells were exposed to PMN myeloper-oxidase (MPO), chloride and glucose plus glucose oxidase (GO) as a hydrogen peroxide (H2O2) generating system. The toxic oxygen species produced by this system caused lysis of the epithelial targets which was dependent on the duration of incubation and the concentrations of MPO and GO. Azide, an inhibitor of MPO, and catalase, a scavenger of H2O2, inhibited the lytic activity of this system. Scanning electron micrographs of gingival epithelial cells cocultured with activated PMNs showed lifting of the cells from the plating surface, while target cells attacked by the MPO system revealed extensive damage of cell membranes. These studies indicate that activated PMNs cause nonlytic detachment injury to gingival epithelial cells which may be mediated by digestion of their extracellular matrix by granule neutral proteases. Furthermore, PMN MPO is capable of generating toxic oxygen species which can lyse these epithelial cells. Collectively, these actions could have profound adverse effects on the function and integrity of the gingival epithelium.

A 43-year-old homosexual man visited his dentist with painful, nodular, ulcerated lesions on the soft palate, right buccal mucosa, and right posterior maxillary gingiva. Serologic studies for exposure to human immunodeficiency virus, performed before biopsy, were positive. Biopsy of the maxillary gingiva demonstrated sheets of histiocytes containing small intracellular yeasts, which on culture were identified as Hisfoplasma capsulstum. Bilateral leukoplakic lesions with some vertical furrowing involving the lateral borders of the tongue were also noted. Histologically, hyperkeratosis and fungal hyphae were identified. The patient was treated for histoplasmosis with amphotericin B, which resulted in significant improvement of the oral lesions. He was subsequently hospitalized for fatigue and dyspnea and was found to have fneumocysfis carinii pneumonia. Pulmonary status deteriorated within a 3-week period, and the patient died. Autopsy findings were negative for histoplasmosis but positive for necrotizing and cavitary P. carinii pneumonia, pulmonary and hepatic herpes simplex infections, and pulmonary and intestinal cytomegalovirus infection. (ORAL SURC ORAL MED 70:631-6)

Epithelial ceUls, isolatedfrom a normal dog pancreatic duct, were grown on coUagen-coated culture inserts suspended above a feeder layer of myofibroblasts. The cels were examined by transmission electron microscopy, immunohistochemistry, cytogenetics, and flow cytometry. In addition, the constitutive and agonist-stimulated mucin secretion of these ceUls was studied using a [3HJN-acetyl-D-glucosamine labeling assay, and the stimulation of intracelular cAMP was measured. CeUls grown on inserts with afeeder layer developed into confluent monolayers consisting of strictly polarized columnar epithelial ceUs with prominent microviUi, intercelular junctions, and normal chromosomal characteristics.

Recent evidence suggests that loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene plays a role in colorectal tumorigenesis and other cancers. However, little is known as to whether the APC gene contributes to the pathogenesis of oral squamous cell carcinoma. To assess involvement of both the APC gene and the human papillomavirus (HPV) in the development of oral pre-malignant and malignant lesions, we analysed DNA from 14 paired oral normal and pre-malignant or malignant paraffin-embedded biopsy specimens, and DNA from cultured normal and HPV 16-immortalised oral epithelial cells for the presence of LOH of APC and for HPV infection, using PCR based techniques. LOH of APC occurred in 80% of cases of oral epithelial dysplasia, 67% of carcinoma in situ, 50% of invasive squamous cell carcinoma cases, and in the HPV 16-immortalised oral epithelial cells. HPV was detected in half of the biopsy specimens, with HPV 16 as the dominant type. More than half of the carcinoma cases were found to contain both LOH of APC and HPV infection. These results suggest that LOH of APC is an early event during oral tumorigenesis. Our findings also suggest a strong correlation between HPV infection, particularly HPV 16, and LOH of the APC gene in oral squamous cell carcinomas.

Chemotherapy is commonly used to treat advanced oral squamous cell carcinoma (SCC) and is known to kill cancer cells through apoptosis. Our hypothesis states that 5-¯uorouracil (5FU) also kills cultured oral epithelial cells through programmed cell death or apoptosis. Cultured oral cancer cells were exposed to an optimum dose of 20 mg/ml of 5FU. Cells were analyzed for changes in cell cycle distribution and induction of cell death including apoptosis. Normal control, human papilloma virusimmortalized (PP), ATCC SCC cell line (CA1) and two primary oral SCC cell lines (CA3 and -4) were studied. Inhibition of apoptosis by a pan-caspase inhibitor was used. SYTO 11¯ow cytometry showed increased apoptosis in all 5FU-treated cell cultures compared to untreated controls. The results show biological variation in apoptotic response. CA1 had the lowest apoptotic rate of the cancer cell lines at 1.5%. Next lowest was CA3, followed by CA4 and PP. In addition, alteration in the G1 and S phase fractions were found. Untreated CA1 showed 28% G1, 53% S compared to 43% G1, and 40% S of treated. We investigated the pathway of apoptosis using the pan-caspase inhibitor IDN-1529 by methylthiazolyl diphenyl tetrazolium bromide (MTT) colorimetric analysis. Results showed mild inhibition of cell death when cells were incubated with 50 mM IDN-1529 for 24 h. This suggests a probable caspase-dependent apoptotic pathway. In conclusion, our data suggest that 5FU induces oral cancer cell death through apoptosis and that biological variation exists between normal and cancer cells and between dierent types of cancer cells themselves. Our data indicate that cultures of a useful in vitro model for chemosensitivity assays are possible. Our results also suggest a caspase-dependent pathway for chemocytotoxicity in oral SCC. #

Attempts to grow human pancreatic duct epithelial cells in long-term culture have proven difficult. We have developed a system of growing these cells for several passages by adapting methods used to culture dog pancreatic duct cells. Epithelial cells were enzymatically dissociated from the main pancreatic duct and plated onto collagen-coated cuhure inserts suspended above a human fibroblast feeder layer. After primary, culture, the cells were either passaged onto new inserts or plastic tissue culture plates in the absence of collagen. Cells grown on the latter plates were maintained in a serum-free medium. Primary pancreatic duct epithelial cells grow steadily to confluence as a monolayer in the feeder layer system. After primary culture, cells passaged onto new inserts with fresh feeder layer or plastic plates and fed with serum-fiee medium continued to develop into confluent monolayers for up to four passages. The cells were columnar with prominent apical microvilli, sub-apical secretory vesicles, and lateral intercellular junctions resembling the morphology of normal in vivo epithelial cells. These ceils were also positive for cytokeratin 19 7, and 8 and carbonic anhydrase II, as measured by immunohistochemistry. Metabolically, these cells synthesized and secreted mucin, as measured by incorporation of tritiated N-acetyl-D-glucosamine. In conclusion, we demonstrated that human pancreatic epithelial cells from the main duct can be successfully grown in culture and repeatedly passagcd using a feeder layer system, with serum-free medium, and in organotypic cultures.

Chemotherapy is commonly used to treat advanced oral squamous cell carcinoma (SCC) and is known to kill cancer cells through apoptosis. Our hypothesis states that 5-¯uorouracil (5FU) also kills cultured oral epithelial cells through programmed cell death or apoptosis. Cultured oral cancer cells were exposed to an optimum dose of 20 mg/ml of 5FU. Cells were analyzed for changes in cell cycle distribution and induction of cell death including apoptosis. Normal control, human papilloma virusimmortalized (PP), ATCC SCC cell line (CA1) and two primary oral SCC cell lines (CA3 and -4) were studied. Inhibition of apoptosis by a pan-caspase inhibitor was used. SYTO 11¯ow cytometry showed increased apoptosis in all 5FU-treated cell cultures compared to untreated controls. The results show biological variation in apoptotic response. CA1 had the lowest apoptotic rate of the cancer cell lines at 1.5%. Next lowest was CA3, followed by CA4 and PP. In addition, alteration in the G1 and S phase fractions were found. Untreated CA1 showed 28% G1, 53% S compared to 43% G1, and 40% S of treated. We investigated the pathway of apoptosis using the pan-caspase inhibitor IDN-1529 by methylthiazolyl diphenyl tetrazolium bromide (MTT) colorimetric analysis. Results showed mild inhibition of cell death when cells were incubated with 50 mM IDN-1529 for 24 h. This suggests a probable caspase-dependent apoptotic pathway. In conclusion, our data suggest that 5FU induces oral cancer cell death through apoptosis and that biological variation exists between normal and cancer cells and between dierent types of cancer cells themselves. Our data indicate that cultures of a useful in vitro model for chemosensitivity assays are possible. Our results also suggest a caspase-dependent pathway for chemocytotoxicity in oral SCC. #