Christopher M. Weghorst

Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and oral squamous cell carcinoma

The frequencies of overexpression and mutation in the p53 tumor suppressor gene were examined in proliferative verrucous leukoplakia and oral squamous cell carcinoma with immunohistochemistry and single-strand conformation polymorphism analysis of DNA fragments amplified by polymerase chain reaction. Ten samples each of normal oral mucosa, proliferative verrucous leukoplakia, and squamous cell carcinoma were immunostained with antibodies against p53 protein; 8 of 10 cases of proliferative verrucous leukoplakia cases and 7 of 10 cases of oral squamous cell carcinoma were positive for p53 protein. Minimal staining was observed in normal oral tissues. The quantified labeling indexes demonstrated a range that corresponded to lesion progression. Single-strand conformation polymorphism analysis revealed p53 gene mutations within exons 5 to 8 in 40% (4 of 10) of the squamous cell carcinoma samples. Two of the 4 mutated squamous cell carcinoma samples lacked p53 expression. No p53 mutations were detected in proliferative verrucous leukoplakia tissues. Human papillomavirus 16 was identified in 2 of 7 p53 positive oral squamous cell carcinoma samples. Human papillomavirus 16 and 18 were identified in two of eight p53 positive proliferative verrucous leukoplakia samples. One p53 negative squamous cell carcinoma sample was positive for human papillomavirus 16 and had a mutation in exon 6 of the p53 gene. Human papillomavirus infection along with p53 expression plays a yet to be defined role in the pathogenesis of a limited number of cases of proliferative verrucous leukoplakia and squamous cell carcinoma. p53 immunohistochemistry, p53 gene mutations, and human papillomavirus infection prevalence do not provide a means to differentiate between leukoplakia and carcinoma and do not provide a predictive test for progression of leukoplakia to carcinoma.

Somatic INK4a‐ARF locus mutations: A significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck

The INK4a‐ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor‐suppressor genes. The p16INK4a (p16) tumor‐suppressor gene product is a negative regulator of cyclin‐dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14ARF tumor‐suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G1 arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0–10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14ARF genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single‐stranded conformational polymorphism (SSCP) analysis termed “cold” SSCP. Exons 1α, 1β, and 2 of INK4a/ARF were amplified using intron‐based primers or a combination of intron‐ and exon‐based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1α. No mutations were found in exon 1β. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14ARF proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41%), a microrearrangement (one of 22, 5%), and single nucleotide substitutions (12 of 22, 56%). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin‐dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild‐type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29‐ to 38‐fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14ARF proteins, seems to be a prevalent event in the development of SCCHN. Mol. Carcinog. 30:26–36, 2001.

p16 Mutation Frequency and Clinical Correlation in Head and Neck Cancer

The tumor suppressor gene p16, when altered, has been shown to play a role in oncogenesis in many different tumor types including head and neck cancer. The goal of this study was to analyse alterations to p16 in squamous cell carcinoma (SCC) of the head and neck and to correlate these with clinical outcome. RNA was isolated from 26 SCC head and neck tumors and from 24 matched controls. A reverse transcription polymerase chain reaction was utilized to generate p16 cDNA, which was sequenced and analysed for alterations. In the 26 patient group 58% of the tumors had a p16 alteration, which were characterized by: 8 deletions, 1 insertion/deletion, 4 point mutations and 2 with no p16 expression. In 24 matched normal tissue samples there were no p16 alterations. Those patients with p16 alterations appear to have survival rates comparable to those without p16 alterations, although patients with p16 alterations appear to have more recurrences.

Anticarcinogenic effects of cadmium in B6C3F1 mouse liver and lung

The B6C3F1 mouse liver has been widely used for the evaluation of carcinogenic or tumor promoting efficacy of various organic compounds, although little is known about the actions of metallic carcinogens in this system. Thus, the ability of cadmium to initiate or promote tumors in B6C3F1 mouse liver was studied. In promotion studies, diethylnitrosamine (DEN; 90 mg/kg, ip) was given as an initiator to 5-week-old mice followed 2 weeks later by 500 or 1000 ppm of cadmium in drinking water for 50 weeks. DEN caused an elevation of liver tumor incidence (13 tumor bearing mice/45 total) over control (1/48) which was prevented by cadmium (DEN + 500 ppm cadmium, 3/42; DEN + 1000 cadmium, 0/47). Cadmium alone did not further reduce the very low spontaneous liver and lung tumor incidence at approximately 1 year of age. DEN-induced lung tumor incidence (15/45) was also reduced by cadmium (DEN + 500 ppm cadmium, 11/42; DEN + 1000 ppm cadmium, 1/47) to control levels (0/48). In initiation studies, cadmium (20 or 22.5 mumol/kg, sc) was given to 5-week-old mice (n = 30-60) 2 weeks before an established promoting regimen of sodium barbital (BB) in drinking water at 500 ppm level was begun. Barbital in drinking water was given continuously for up to 92 weeks. Such cadmium doses caused acute, focal hepatic necrosis. Mice treated with BB and killed at 97 weeks of age showed an elevation of liver tumor multiplicity (7.44 tumors/liver) over control (2.24) that was prevented by cadmium in a dose-related manner (20 mumol/kg cadmium + BB, 3.93; 22.5 mumol/kg cadmium + BB, 1.87). Cadmium alone given by injection also reduced spontaneous liver tumor multiplicity. These results indicate that cadmium inhibits tumor formation in the B6C3F1 mouse liver initiation/promotion system regardless of route of exposure or sequence of administration. The possibility exists that cadmium has a specific toxicity toward previously initiated cells within liver and lung.

Frequent Alterations of p16INK4a and p14ARF in Oral Proliferative Verrucous Leukoplakia

Proliferative verrucous leukoplakia (PVL) represents a rare but highly aggressive form of oral leukoplakia with >70% progressing to malignancy. Yet, PVL remains biologically and genetically poorly understood. This study evaluated the cell cycle regulatory genes, p16INK4a and p14ARF, for homozygous deletion, loss of heterozygosity, and mutation events in 20 PVL cases. Deletion of exon 1β, 1α, or 2 was detected in 40%, 35%, and 0% of patients, respectively. Deletions of exons 1α and 1β markedly exceed levels reported in non-PVL dysplasias and approximate or exceed levels reported in oral squamous cell carcinomas. Allelic imbalance was assessed for markers reported to be highly polymorphic in squamous cell carcinomas and in oral dysplasias. Loss of heterozygosity was detected in 35.3%, 26.3%, and 45.5% of PVLs for the markers IFNα, D9S1748, and D9S171, respectively. INK4a and ARF sequence alterations were detected in 20% and 10% of PVL lesions, accordingly. These data show, for the first time, that both p16INK4a and p14ARF aberrations are common in oral verrucous leukoplakia; however, the mode and incidence of inactivation events differ considerably from those reported in non-PVL oral premalignancy. Specifically, concomitant loss of p16INK4a and p14ARF occurred in 45% of PVL patients greatly exceeding loss reported in non-PVL dysplastic oral epithelium (15%). In addition, p14ARF exon 1β deletions were highly elevated in PVLs compared with non-PVL dysplasias. These data illustrate that molecular alterations, even within a specific genetic region, are associated with distinct histologic types of oral premalignancy, which may affect disease progression, treatment strategies, and ultimately patient prognosis. (Cancer Epidemiol Biomarkers Prev 2008;17(11):3179–87)

The p16/Cdkn2a/Ink4a Gene Is Frequently Deleted in Nitrosourea-Induced Rat Glial Tumors

The present study investigates nitrosourea-induced rat (Rattus norvegicus) glioma cell lines for the functional status of the p16/Cdkn2a/Ink4a gene, which encodes the p16 cdk4 inhibitor and the alternative reading frame protein, p19ARF. We detected homozygous deletions of the p16/Cdkn2a/Ink4a gene locus in 4 of 5 glioma cell lines (C6, F98, RG2, and RGL.3), but not in the 9L gliosarcoma cell line or in a rat primary fibroblast cell line. RT-PCR demonstrated expression of the p16 and p19ARF mRNAs only in 9L cells and in rat fibroblasts. Comparative genomic in situ hybridization showed that the copy number of rat chromosome RNO5 was not altered in any of the glioma cell lines investigated, indicating that the deletions result from a discrete loss in the region of the p16/Cdkn2a/Ink4a locus. This is the first report of p16/Cdkn2a/Ink4a deletions present in nitrosourea-induced rat glioma cell lines. Since this genetic alteration is also commonly observed in human malignant glial tumors, our results validate the use of chemically induced rat glioma cell lines as an experimental model in the development of gene therapy strategies.

Allelic imbalance in oral lichen planus and assessment of its classification as a premalignant condition

OLP is a relatively common immune-mediated mucosal condition with a predilection for middle-aged women. Although classified as a premalignant condition, this classification remains controversial. Using stringent diagnostic criteria, some authors have found that OLP patients are not at increased risk for oral SCC. Credible but limited genetic evidence also indicates that epithelial tissues from OLP patients diagnosed using stringent criteria differs from premalignant or malignant oral lesions but is similar to epithelium from benign oral lesions. To further investigate this genetic line of evidence, biopsy specimens diagnosed as fibroma, OLP, low-grade dysplasia, high-grade dysplasia, and SCC were retrieved from the archives of the Oral Pathology Consultants at the Ohio State University. Using laser capture microdissection, tissue of interest was captured from each case and DNA subsequently extracted. Fluorescently labeled PCR primers were used to amplify DNA at 3 tumor suppressor gene loci (3p14.2, 9p21, and 17p13) and evaluated for LOH or microsatellite instability (MSI). OLP was found to be significantly different from low-grade dysplasia, high-grade dysplasia, and SCC when LOH/MSI was found at more than 1 loci (P = .011, P = .032, P = .003), but not different from benign fibromas (P = .395). In agreement with previous studies, well-documented cases of OLP diagnosed using stringent criteria exhibit a genetic profile more similar to a benign or reactive process than a premalignant/malignant one. These findings do not support the classification of OLP as a premalignant condition.

Expression of Cell Cycle Proteins in 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone-Induced Mouse Lung Tumors

Cyclin D1 dysregulation and differential inactivation of p16INK4a and Rb have been observed in human lung cancer. In chemically induced mouse lung tumors, the p16INK4a gene is a target of inactivation, and Rb is reduced at the mRNA level (Northern blot) although similar at the protein level (Western blot) when compared to normal lung tissues. The expression of cyclin D1, cdk4, p16INK4a, and Rb protein was examined by immunohistochemistry in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumors. Immunohistochemical staining revealed exclusive nuclear staining of both cyclin D1 and cdk4 that was light to moderate in normal mouse lung tissues, but intense in lung adenomas and adenocarcinomas. Western blot analysis confirmed the increased expression of cyclin D1 and cdk4 in lung tumors compared to normal lung. Immunohistochemical analyses of lung tumors showed focal areas which lacked p16INK4a staining. Expression of p16INK4a, as determined by RT-PCR, was variable in lung tumors. Mutations in p16INK4a were not found by SSCP analysis. Immunohistochemical analyses of normal lung tissues showed intense staining for Rb protein in alveolar epithelial cells and in other lung cell types; however, in the lung tumors the staining intensity was reduced and the distribution was altered. Expression of Rb was detected in normal lung tissues but was barely detectable by Northern blot hybridization in lung tumors. Western blot analysis indicated the presence of both hypophosphorylated and hyperphosphorylated Rb protein in lung tumors and in normal lung tissues. These results suggest that alterations in the cell cycle proteins, cyclin D1, cdk4, p16INK4a, and Rb, may play a role in the acquisition of autonomous growth by adenomas. Furthermore, they demonstrate the importance of immunohistochemical studies to examine expression in tissues that contain multiple cell types, such as the lung, and in tumors that by nature are heterogeneous.

A black raspberry extract inhibits proliferation and regulates apoptosis in cervical cancer cells

OBJECTIVE Cervical cancer is the second most common female cancer worldwide, and it remains a challenge to manage preinvasive and invasive lesions. Food-based cancer prevention entities, such as black raspberries and their derivatives, have demonstrated a marked ability to inhibit preclinical models of epithelial cancer cell growth and tumor formation. Here, we extend the role of black raspberry-mediated chemoprevention to that of cervical carcinogenesis. METHODS Three human cervical cancer cell lines, HeLa (HPV16-/HPV18+, adenocarcinoma), SiHa (HPV16+/HPV18-, squamous cell carcinoma) and C-33A (HPV16-/HPV18-, squamous cell carcinoma), were treated with a lyophilized black raspberry ethanol extract (RO-ET) at 25, 50, 100 or 200μg/ml for 1, 3 and 5days, respectively. Cell proliferation was measured by WST1 (tetrazolium salt cleavage) assays. Flow cytometry (propidium iodide and Annexin V staining) and fluorescence microscopy analysis were used to measure apoptotic cell changes. RESULTS We found that non-toxic levels of RO-ET significantly inhibited the growth of human cervical cancer cells, in a dose-dependent and time-dependent manner to a maximum of 54%, 52% and 67%, respectively (p<0.05). Furthermore, cell growth inhibition was persistent following short-term withdrawal of RO-ET from the culture medium. Flow cytometry and fluorescence microscopy demonstrated RO-ET-induced apoptosis in all cell lines. CONCLUSION Black raspberries and their bioactive components represent promising candidates for future phytochemical-based mechanistic pathway-targeted cancer prevention strategies.

Identification and sequencing of the Syrian Golden hamster (Mesocricetus auratus) p16INK4a and p15INK4b cDNAs and their homozygous gene deletion in cheek pouch and pancreatic tumor cells

Previous studies have shown that the p16(INK4a) tumor suppressor gene is inactivated in up to 98% of human pancreatic cancer specimens and 83% of oral squamous cell carcinomas. Inactivation of the related p15(INK4b) gene has also been identified in a number of tumors and cell lines, however, its role as an independent tumor suppressor remains to be elucidated. Chemically-induced tumors in the Syrian Golden hamster (Mesocricetus auratus) have been shown to be excellent representative models for the comparative development and progression of a number of human malignancies. The purpose of this study was to determine the importance of the p16(INK4a) and p15(INK4b) genes in two experimental hamster models for human pancreatic and oral carcinogenesis. First, hamster p16(INK4a) and p15(INK4b) cDNAs were cloned and sequenced. The hamster p16(INK4a) cDNA open reading frame (ORF) shares 78%, 80%, and 81% identity with the human, mouse, and rat p16(INK4a) sequences, respectively. Similarly, the hamster p15(INK4b) cDNA ORF shares 82% and 89% sequence identity with human and mouse p15(INK4b), respectively. Second, a deletion analysis of hamster p16(INK4a) and p15(INK4b) genes was performed for several tumorigenic and non-tumorigenic hamster cell lines and revealed that both p16(INK4a) and p15(INK4b) were homozygously deleted in a cheek pouch carcinoma cell line (HCPC) and two pancreatic adenocarcinoma cell lines (KL5B, H2T), but not in tissue matched, non-tumorigenic cheek pouch (POT2) or pancreatic (KL5N) cell lines. These data strongly suggest that homozygous deletion of the p16(INK4a) and p15(INK4b) genes plays a prominent role in hamster pancreatic and oral tumorigenesis, as has been well established in correlative studies in comparable human tumors. Furthermore, this study supports the comparative importance of the hamster pancreatic and cheek pouch models of carcinogenesis in subsequent mechanistic-, therapeutic-, and preventive-based studies aimed at providing important translational data applicable to pancreatic adenocarcinoma and oral squamous cell carcinoma in humans.