One of the most important components of G1 checkpoint is the retinoblastoma protein (pRB 110 ). The activity of pRB is regulated by its phosphorylation, which is mediated by genes such as cyclin D1 and p16 / MTS1 . All three genes have been shown to be commonly altered in human malignancies. We have screened a panel of 26 oral squamous cell carcinomas (OSCC), nine premalignant and three normal oral tissue samples as well as eight established OSCC cell lines for mutations in the p16 / MTS1 gene. The expression of p16 / MTS1 , cyclin D1 and pRB 110 was also studied in the same panel. We have found p16 / MTS1 gene alterations in 5/26 (19%) primary tumours and 6/8 (75%) cell lines. Two primary tumours and five OSCC cell lines had p16/MTS1 point mutations and another three primary and one OSCC cell line contained partial gene deletions. Six of seven p16/MTS1 point mutations resulted in termination codons and the remaining mutation caused a frameshift. Western blot analysis showed absence of p16/MTS1 expression in 18/26 (69%) OSCC, 7/9 (78%) premalignant lesions and 8/8 cell lines. One cell line, H314, contained a frameshift mutation possibly resulting in a truncated p16/MTS1 protein. pRB was detected in 14/25 (56%) of OSCC but only 11/14 (78%) of these contained all or some hypophosphorylated (active) pRB. In premalignant samples, 6/8 (75%) displayed pRB, and all three normal samples and eight cell lines analysed contained RB protein. p16/MTS1 protein was undetectable in 10/11 (91%) OSCCs with positive pRB. Overexpression of cyclin D1 was observed in 9/22 (41%) OSCC, 3/9 (33%) premalignant and 8/8 (100%) of OSCC cell lines. Our data suggest p16/MTS1 mutations and loss of expression to be very common in oral cancer cell lines and less frequent in primary OSCC tumours. A different pattern of p16/MTS1 mutations was observed in OSCC compared to other cancers with all the detected p16/MTS1 mutations resulting in premature termination codons or a frameshift. The RB protein was expressed in about half (44%) of OSCCs and its expression inversely correlated with p16/MTS1 expression. In conclusion, we show that abnormalities of the RB pathway are a common mechanism of oral carcinogenesis. © 1999 Cancer Research Campaign

Lysyl oxidase (LO) takes part in the initial steps of converting soluble monomers of collagen and elastin into insoluble fibres in the extracellular matrix. We have studied the immunolocalization of LO as a marker of fibrogenesis in oral submucous fibrosis (OSF). Oral biopsies from 13 subjects with OSF, 6 with histologically confirmed squamous cell carcinoma (SCC) arising in OSF and 10 SCC non‐related to OSF, were examined. Strong positive staining was observed in 7/13 OSF samples in the cytoplasmic processes of fibroblasts and extracellularly in the upper third of the lamina propria. Furthermore, LO was found to co‐localize in the areas stained strongly for collagen and elastin by histochemical stains. Examination of SCC tissues showed localization of LO adjacent to invading epithelial islands as evidence of a stromal reaction both in carcinomas arising from OSF and in SCC from non‐OSF cases. These findings suggest that upregulation of LO may be an important factor in the pathogenesis of OSF and in the early stromal reaction of oral cancer.

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Summary Forty-nine ovarian tumours were examined for loss of heterozygosity (LOH) on chromosome 5 using eight microsatellite markers spanning both arms, including one at the APC locus. LOH on Sq was a frequent event, detectable in 23 of 49 (47%) tumours, whereas 5p LOH was detected in only 1 of 22 tumours (5%). Six tumours showed partial LOH on 5q, enabling the candidate region to be localised to a 22 cM region proximal to APC, flanked by D5S424 and D5S644. An association was found between 5q LOH and TP53 mutation, with 18 of 23 (78%) tumours with LOH on Sq also harbouring a TP53 mutation. LOH on Sq was observed in 6 of 18 (33%) stage I tumours, suggesting that it may be an early event in the molecular pathogenesis of certain ovarian carcinomas.

Loss of CDKN2A expression was demonstrated by immunohistochemistry in 87% of oral and oropharyngeal squamous cell carcinoma (OSCC) primary tumor samples. By contrast, DNA studies showed a much lower frequency of loss of the CDKN2A gene. Point mutations and promoter methylation of CDKN2A were seen in 7% and 23%, respectively, of primary tumors. Loss of heterozygosity analysis using a dense set of 9p markers showed allelic imbalance that included CDKN2A in only 31% of samples, but a further 47% showed loss at loci near CDKN2A with apparent retention of CDKN2A. No tumor with any allelic imbalance expressed CDKN2A, whether or not the imbalance appeared to involve the CDKN2A locus. We interpret these data as showing partially overlapping deletions on the two 9p homologues, with homozygous deletion of CDKN2A masked by amplification of contaminating stromal material. Our data show that inactivation of the CDKN2A gene products is a near-universal step in the development of oral and oropharyngeal squamous cell carcinomas, and we suggest that homozygous deletion is the most common mechanism of inactivation. The CDKN2A locus may be particularly prone to deletion because it encodes two unrelated tumor suppressor proteins, CDKN2A (p16INK4a) and p19ARF, and deletion, but not point mutation or methylation, would inactivate both gene products. However, our results also suggest that complex patterns of allelic imbalance in primary squamous carcinomas in general may not provide reliable evidence for the existence of multiple tumor suppressor genes within a single chromosomal region.

Aberrations of the p53 gene and the overexpression of its protein are described in a variety of neoplasms, including oral and other head and neck cancers. Here we report the association of p53 (over)expression with a downstream cell cycle inhibitor p21/waf 1 in oral squamous cell carcinoma (SCC). The loss of expression of p16 and p27, two other cyclin‐dependent kinase (cdk) inhibitors, was also examined. In this panel of tumours, 10/24 carcinomas were p53‐immunopositive. Heterogeneous expression of p21 and p27 was seen in 10/24 SCC and 9/16 SCC, respectively, and this was not correlated to p53 status. The expression of p21 and p27 in these SCCs suggests the existence of mechanisms by which some growing tumour cells may tolerate these cell cycle inhibitors; eight SCCs lacked expression of both inhibitors but only two of these cancers overexpressed p53, suggesting that accumulation of p21/p27 can be independent of the functional status of the p53 gene. Data do not support a clear example of a phenotype that shows an overexpression of p53 with downregulation of p21 or p27 leading to cell cycle alterations. Furthermore, only three SCCs were p16‐negative and p53‐positive. This suggests that these two tumour suppressors may act in separate pathways.

Trivedy C, Warnakulasuriya KAAS, Tavassoli M, Steingrimsdottir H, Penhallow J, Maher R, Johnson NW: p53 aberrations in oral submucous fibrosis and oral squamous cell carcinoma detected by immunocytochemistry and PCR‐SSCP. J Oral Pathol Med 1998; 27: 72–7. © Munksgaard, 1998. An archival series of oral biopsies from Karachi, Pakistan, consisting of 21 cases of oral submucous fibrosis (OSF) and 27 cases of squamous cell carcinoma (SCC), of which 6 had arisen from OSF, were used to examine the aberrations in the structure and expression of the p53 tumour suppressor gene. The PCR‐SSCP method was used for mutation analysis of exons 2–9, and (over)expression of p53 protein was detected by immunocytochemistry using monoclonal antibody DO 7. Positive immunostaining was observed in 15/20 (75%) of OSF specimens, 3/6 (50%) of SCC arising from OSF and 14/21 (67%) of SCC not arising from OSF. Mobility shifts in SSCP indicative of a mutation in p53 or loss of heterozygosity (deletion of a band) were seen in 13/21 cases of OSF and 15/27 cases of SCC. There was concordance between immunocytochemistry and SSCP results in a majority (33/48) of samples. Though the number of analysed SCC cases arising from OSF was limited, the results suggest that p53 mutation/protein stabilisation may play a part in the pathogenesis of OSF and its progression to SCC.

Integration of proviral DNA into the host cell genome is a characteristic feature of the retroviral life cycle. This process involves coordinate DNA strand break formation and rejoining reactions. The full details of the integration process are not yet fully understood. However, the endonuclease and DNA strand-joining activities of the virus-encoded integrase protein (IN) are thought to act in concert with other, as-yet-unidentified, endogenous nuclear components which are involved in the DNA repair process. The nuclear enzyme poly(ADPribose) polymerase (PARP), which is dependent on DNA strand breaks for its activity, is involved in the efficient repair of DNA strand breaks, and maintenance of genomic integrity, in nucleated eukaryotic cells. In the present work, we examine the possible involvement of PARP in the retroviral life cycle and demonstrate that inhibition of PARP activity, by any one of three independent mechanisms, blocks the infection of mammalian cells by recombinant retroviral vectors. This requirement for PARP activity appears to be restricted to processes involved in the integration of provirus into the host cell DNA. PARP inhibition does not affect viral entry into the host cell, reverse transcription of the viral RNA genome, postintegration synthesis of viral gene products, synthesis of the viral RNA genome, or the generation of infective virions. Therefore, efficient retroviral infection of mammalian cells is blocked by inhibition of PARP activity.