HPV-16, tobacco-specific N-nitrosamine, and N-methyl-N'-nitro-N-nitrosoguanidine in oral carcinogenesis.

We previously immortalized human oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines. These transfected cells were morphologically different from the normal counterpart, contained intact HPV-16 DNA in an integrated form, and expressed numerous viral genes. These cells contained lower levels of wild-type p53 protein and higher levels of c-myc mRNAs compared to normal cells. However, they proliferated only in keratinocyte growth medium containing a low level of calcium and were not tumorigenic in nude mice. A HPV-16-immortalized cell line was exposed to either 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N-methyl-N'-nitro-N-nitrosoguanidine. Four chemically transformed cell colonies were isolated. These cells proliferated well in Dulbecco's minimum essential medium containing a physiological level of calcium. They contained, similar to the immortalized counterpart, integrated HPV-16 sequences and lower levels of both wild-type p53 protein and DCC messages compared to normal cells. Among the chemically transformed cells, two colonies obtained from 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone exposure demonstrated an enhanced proliferation capacity in nude mice and transcribed a substantially higher amount of HPV-16 E6/E7, epidermal growth factor receptors, and c-myc genes compared with the immortalized counterpart. These experiments indicate that malignant transformation of oral keratinocytes can be caused by a sequential combined effect of "high risk" HPV and tobacco-related carcinogens.

Effect of UV-irradiation on cell cycle, viability and the expression of p53, gadd153 and gadd45 genes in normal and HPV-immortalized human oral keratinocytes.

We previously demonstrated neoplastic conversion of HPV-immortalized human oral keratinocytes by exposing cells to chemical carcinogens, but failed to transform normal human oral keratinocytes with same chemical carcinogens in vitro. Though the reason for different responses of normal and HPV-immortalized oral keratinocytes to chemical carcinogens remains speculative, the difference may be due to the capacity of normal cells and incapacity of HPV-immortalized cells for repairing damaged DNA induced by carcinogens. Since (1) the repair of damaged DNA takes place in G1/G2 phases of cell cycle, (2) wild type p53 plays major role in the induction of transient G1 and/or G2 arrests, and (3) the expression of gadd45 and gadd153 is also associated with the cell cycle arrest and DNA damage, we investigated transient cell cycle arrest and the expression of p53, gadd45 and gadd153 in normal human oral keratinocytes, HPV-immortalized oral keratinocytes, and an oral cancer cell line expressing mutant p53 after exposing cells to UV light. Normal cells demonstrated transient G1 arrest after exposure to UV light, but other tested cells did not. While UV-irradiation significantly increased the level of intranuclear wild type p53 protein in normal cells, it did not alter p53 protein levels in HPV-immortalized and oral cancer cells. The level of gadd45 transcripts was enhanced in all tested cells, but normal cells demonstrated higher increase in the level of gadd45 after UV-exposure compared to other tested cells. The level of gadd153 gene transcripts was only increased in normal oral keratinocytes after UV-irradiation. These data indicate that UV-induced transient G1 arrest in normal oral keratinocytes may be associated with both enhanced levels of intranuclear wild type p53 protein and gadd45 and gadd153 transcripts.

Combined oral carcinogenicity of HPV-16 and benzo(a)pyrene: an in vitro multistep carcinogenesis model.

We previously immortalized normal human oral keratinocytes by transfection with recombinant HPV-16 DNA and subsequently exposed the cells to benzo(a)pyrene for 7 days. The exposure to benzo(a)pyrene modified the immortalized cells: the modified cells (HOK-16B-BaP) proliferated in an ordinary culture medium containing physiological calcium level (1.5 mM), but demonstrated only enhanced proliferation capacity without tumor formation in nude mice and failed to show in vitro anchorage-independency. In this study, we further modified the HOK-16B-BaP cells by subculturing the cells in a medium containing benzo(a)pyrene for 6 months. The cells were further modified with a chronic benzo(a)pyrene exposure and were termed HOK-16B-BaP-T cells (1) demonstrated a malignant phenotype in organotypic 'raft' culture, (2) showed in vitro anchorage-independency, (3) developed tumors in nude mice when injected subcutaneously, (4) contained a significantly higher copy number of intact and integrated HPV-16 DNA; (5) contained higher level of HPV-16 E6/E7 messages and E7 protein, (6) were more resistant to transforming growth factor-beta 1 and (7) secreted higher level of vascular endothelial growth factor with molecular weight of 56 kd than parental HOK-16B-BaP cells. However, the levels of p53 and ras proteins and the levels of p53, c-myc and c-fos transcripts in the HOK-16B-BaP-T cells were not different from those in the HOK-16B-BaP cells. The highly conserved coding regions of the p53, c-Ha-ras1, and c-Ki-ras2 genes of the tumor cells were not mutated. These data indicate that the HPV-immortalized human oral keratinocytes can convert to tumorigenic cells by chronic exposure to benzo(a)pyrene. The tumorigenic conversion seems to be associated with (1) the overexpression of viral oncogenes such as E6 and E7 genes, (2) the higher resistance of cells to transforming growth factor-beta 1 and (3) the high secretion of 56 kd vascular endothelial growth factor from the cells.

Peripheal blood toxicity of topical 5-fluorouracil on the Syrian hamster cheek pouch.

Toxicological studies on the peripheral blood after topical application of 5-fluorouracil to the buccal pouch of the Suriam hamster showed that a daily application of 10 mul (9.5 mg) was the maximum ideal dose for this animal. This dose did not depress to a significant degree the white blood cell count, red blood cell count, hemoglobin leve., hematocrit value, or the peripheral differential blood counts after 14 daily applications. Doses of increased concentration either daily or periodically were less effective and toxic.

State of p53, Rb and DCC tumor suppressor genes in human oral cancer cell lines.

The tumor suppressor genes p53, Rb, and DCC were studied in five human oral cancer cell lines (FaDu, SCC-4, HEp-2, 1483, and OEC-M1) and in primary normal human oral keratinocytes (NHOK). All tested cancer lines had similar amount of p53 messages to normal cells, but the cancer lines FaDu and SCC-4 contained significantly higher p53 protein levels than did the normal counterpart. Sequencing p53 cDNA for these cancer cells showed point mutations: In the FaDu cell line, a mutation of CGG to CTG occurred at codon 248; and in the SCC-4 cell line, a mutation of CCC to TCC occurred at codon 151. The HEp-2 and 1483 cancer lines translated very low levels of p53 protein compared to the normal counterpart. Sequencing of p53 cDNA for HEp-2 and 1483 lines showed no mutations. Southern and Northern analyses revealed that these cell lines harbored HPV-18 DNA and expressed the viral E6/E7 protein. The OEC-M1 line showed different restriction fragment length polymorphism for the p53 gene compared with other cells, and did not express p53. All oral cancer cell lines except the OEC-M1 cells expressed both phosphorylated and hypophosphorylated Rb proteins. Further, the OEC-M1 line expressed smaller sized hypophosphorylated Rb proteins compared with normal cells. Unlike the other cancer lines, the HEp-2 and OEC-M1 lines also did not contain DCC mRNAs. These data indicate that "high risk" HPV infections and mutations of p53, Rb, and DCC genes are frequently found in oral cancer cells and may be associated with oral cancer.

Preparation and evaluation of a nonproprietary bilayer skin substitute.

Cross-linked, allogeneic, telopeptide-depleted dermal grafts were lyophilized and laminated with silicone rubber elastomer. Resultant bilayers were studied for incorporation into the wound site and capacity to inhibit cutaneous wound contraction in experimental animals. Bilateral full-thickness skin wounds were made in 20 male New Zealand white rabbits. One side was grafted with the processed graft, while the contralateral side remained ungrafted as a control wound. Over 63 days, wound sites were analyzed at intervals on the basis of the extent and rate of wound contraction and by histologic examination. Cutaneous wounds successfully incorporated graft matrix and were significantly inhibited in their rate and extent of wound contraction. Notably, by day 63, grafted wounds retained 71 percent of their original area, whereas ungrafted control wounds retained only 16 percent of their original area. There were no graft rejections, and the bilayer graft's dermal analogue appeared to function as a biodegradable template that physically conformed neodermis to a preestablished pattern while counteracting contractile forces. This investigation suggests that, in experimental animals, the success of bilayer dermal grafts is less dependent on highly specialized and complex preparative techniques than typically has been presumed and that relatively simple, previously published, nonproprietary techniques, when adapted to a bilayer format, yield acceptable results as defined in terms of biocompatibility, capacity for graft incorporation, and inhibition of wound contraction.

Rhinocerebral mucormycosis.

Rhinocerebral mucormycosis is a fungal diseases that has a 50% mortality. Its occurrence has increased, possibly because of greater use of chemotherapeutic agents that mya compromise the immunologic defenses of the host or alter the normal flora. The earliest signs, ulceration and pain, may appear in the mouth. In the patient described in this report, the autopsy showed that mucormycosis had entered the brain cells.

Glycogen-rich adenocarcinoma of minor salivary glands. A light and electron microscopic study.

A malignant glycogen-rich adenocarcinoma of palatal salivary glands is reported. Histopathology revealed nonencapsulated nests and cords of polyhedral cells showing voluminous clear cytoplasms and cellular pleomorphisms, separated by fine vascular septae. Small and large ducts were infrequently seen showing apparent transition of large ducts into clear cells. The tumor cells were PAS- and Best-carmine positive, diastase soluble, and mucicarmine and Alcian-blue negative. Ultrastructurally, the tumor cells were arranged in solid nests and cords of electron-lucent cells surrounding single or multiple lumina, and surrounded by basement lamina. Occasional fusiform electron-dense cell processes were present beneath the basement lamina. The electron-lucent cells were joined by junctional complexes, contained intracytoplasmic canals, and were filled with massive accumulations of beta glycogen particles. The electron-dense processes contained interlacing whorls of fine filaments and exhibited peripheral focal densities. The findings suggest that this glycogen-rich malignant tumor is of epithelial origin most probably of ductal cells.

Effect of snuff extract on the replication and synthesis of viral DNA and proteins in cells infected with herpes simplex virus.

The water-extractable component of snuff (snuff extract) inhibits the replication of herpes simplex virus (HSV) by suppressing the synthesis of viral DNA. This process probably causes HSV to be oncogenic. To further understand the mechanism of inhibitory action of snuff extract on HSV replication, the effect of snuff extract on the synthesis of viral DNA and proteins in type 1 HSV (HSV-1) infected cells was investigated. Snuff extract inhibited the synthesis of viral DNA and altered the production of certain classes of viral proteins. The syntheses of ICP4, a viral alpha-protein, and ICP8, a beta-protein, were not generally reduced by noncytotoxic concentrations of snuff extract (where ICP = infected cell polypeptide). However, snuff extracts significantly inhibited the production of ICP gC (glycoprotein C), a gamma 2-protein, and the inhibition was in a concentration-dependent fashion: the higher the concentration of snuff extracts, the greater the inhibition. Based on the fact that the production of alpha- and beta-proteins is absolutely necessary for and precedes the viral DNA synthesis and that viral gamma 2-proteins are mostly produced by the newly synthesized viral DNA, it is concluded that snuff extract inhibits HSV-1 DNA replication directly rather than indirectly via the alteration of viral protein synthesis.

Intraoral juvenile xanthogranuloma.

A case of juvenile xanthogranuloma of the gingiva is presented. This uncommon, benign disorder has rarely been histologically documented in the oral cavity, and rarely have oral lesions been described as presenting symptoms prior to this report. The pertinent literature is reviewed and possible etiologic factors are discussed.

Effect of smokeless tobacco and tobacco-related chemical carcinogens on survival of ultraviolet light-inactivated herpes simplex virus.

Low doses of ultraviolet (UV) light, x-rays, photodynamic treatment, or aflatoxins increase the survival of UV-irradiated virus in cells. This effect is postulated to occur by enhancement of the error-prone cellular repair function, which could also be associated with oncogenic cell transformation. The present study was designed to investigate whether treatment of green monkey kidney cells with water extract of snuff (snuff extract), benzo[a]pyrene, nicotine, or tobacco-specific N'-nitrosamines would result in enhanced survival of UV-irradiated herpes simplex virus (HSV). Exposure of the cells with snuff extract, benzo[a]pyrene. N'-nitrosonornicotine, or 4-(N-methyl-N'-nitrosamino)-1-(3-pyridyl)-1-butanone resulted in an enhancement of survival of UV-irradiated HSV type 1 compared with the control whereas exposure of the cells with nicotine did not. These data indicate that the water-extractable component of snuff and tobacco-related chemical carcinogens increase the cellular repair mechanism and provides for increased survival of UV-irradiated HSV.

Anchorage-independent growth and the expression of cellular proto-oncogenes in normal human epidermal keratinocytes and in human squamous cell carcinoma cell lines.

The expression of multiple cellular proto-oncogenes and the in vitro anchorage-independent growth of normal human epidermal keratinocytes and several human squamous cell carcinoma cell lines were studied and correlated. Squamous cell carcinoma cell lines KB, Si Ha, HEp-2, and Fa Du showed high anchorage independency, and MS 751 and A-253 cell lines had minimum independency. However, the normal keratinocytes and the A-431 cell line did not show anchorage-independent growth. Both the normal human epidermal keratinocytes and cancer cell lines expressed multiple proto-oncogenes such as src, erb B-1, abl, fos, raf, H-ras, and myc, and the amount of expression of these oncogenes was notably higher in the cancer cell lines than in the normal keratinocytes. The expression of proto-oncogenes from the monolayer cultures of the cancer cell lines is poorly correlated with the anchorage independency of the cells. These data indicate that the anchorage independency is not directly linked to the expression of specific cellular proto-oncogene(s) of the monolayer cancer cell cultures.

Three cell lines from hamster buccal pouch tumors induced by topical 7,12-dimethylbenz(a)anthracene, alone or in conjunction with herpes simplex virus inoculation.

Three squamous carcinoma cell lines HBPC-1, HBPC-2, and HBPC-3 were established from hamster buccal pouch tumors induced by topical 7,12-dimethylbenz(a)anthracene (DMBA) treatment alone, topical DMBA treatment in conjunction with type 1 herpes simplex virus (HSV-1) inoculation, and topical DMBA application in combination with type 2 HSV (HSV-2) inoculation, respectively. The cells were epithelial in morphology, had a doubling time of approximately 18 h, and required bovine serum for optimal growth. They demonstrated an in vitro anchorage-independent growth and produced squamous cell carcinomas when transplanted into normal hamster pouch submucosa. The carcinoma cell lines equally expressed cellular hst, src, abl, and raf proto-oncogenes that were not expressed in the normal hamster pouch epithelial cells. An equal amount of fos gene expression was noticed in the normal pouch epithelial cells, HBPC-1 and HBPC-3, but the HBPC-2 expressed less fos poly(A+)RNA than the other cell lines. The myc proto-oncogene was also expressed both in the normal pouch epithelial cells and in the cancer cell lines. However, the size and number of expressed myc poly(A+)RNA in the normal cells and cancer cell lines differed. Although the normal cells and HBPC-1 expressed a single myc transcript, 1.7-kilobase (kb) and 2.3-kb, respectively, both HBPC-2 and HBPC-3 expressed two myc poly(A+)RNAs, 1.7-kb and 2.3-kb.

Sequential combined tumorigenic effect of HPV-16 and chemical carcinogens.

We immortalized oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines, human oral keratinocytes-16A (HOK-16A) and -16B (HOK-16B). These cell lines were morphologically different from the normal counterpart, contained HPV-16 DNA as integrated form and expressed numerous viral genes. However, these cells proliferated only in culture medium containing low calcium (0.15 mM) and are not tumorigenic in nude mice. To test the hypothesis that tumors can be developed by sequential combined effect of human papillomavirus and chemical carcinogens in the oral cavity, these immortalized cell lines were chemically transformed by exposure to either benzo[a]pyrene or methanesulfonic acid ethyl ester. Such transformants proliferated in medium containing physiological calcium levels (1.5 mM) and demonstrated enhanced growth potential in nude mice, whereas primary human oral keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. Chemically transformed cells contained integrated, intact HPV-16 sequences and transcribed significantly higher amount of HPV-16 E6/E7 messages and transforming growth factor-alpha (TGF-alpha) compared with the immortalized oral keratinocytes. Like the HPV-immortalized cell lines, the chemically transformed oral keratinocytes contained lower levels of newly synthesized, wild-type p53 proteins compared to normal cells, and expressed wild-type c-Ha-ras. These results indicate that this in vitro system is useful for investigating the mechanisms of multistep oral carcinogenesis.

Active HSV-1 immunization prevents the cocarcinogenic activity of HSV-1 in the oral cavity of hamsters.

Previous investigations have demonstrated that herpes simplex virus (HSV) increased the oral carcinogenic activity of 7,12-dimethylbenz[a]anthracene (DMBA) probably by enhancing the DMBA-induced amplification and overexpression of c-erb-B-1 proto-oncogene in hamster buccal pouch epithelium. The present study investigated the effect of active type 1 HSV (HSV-1) immunization on the development of oral cancer induced by HSV-1 and DMBA, alone or in combination, in the hamster buccal pouch. The results were similar to our previous report in that HSV-1 significantly enhanced the oncogenic effect of DMBA, and the numbers of pouches harboring tumor nodules and the numbers and sizes of tumors developed by topical DMBA were significantly increased by HSV-1 inoculation to the site of the DMBA application. Although HSV-1 immunization did not alter the carcinogenic activity of DMBA in animals receiving topical DMBA in combination with mock inoculation, it prevented the cocarcinogenic effect of HSV-1 in animals receiving topical DMBA in conjunction with HSV-1 inoculation. These data indicate that active HSV-1 immunization completely obstructs the co-oncogenic effect of HSV-1 in the oral cavity of hamsters.