Participation of the nonreducing terminal beta-galactosyl residues of the neutral N-linked carbohydrate chains of porcine zona pellucida glycoproteins in sperm-egg binding.

The zona pellucida (ZP) surrounding the mammalian oocyte is composed of three glycoprotein components (ZPA, ZPB, and ZPC). Mammalian sperm bind to carbohydrate chains of a ZP glycoprotein in the initial phase of fertilization. Sperm-ligand carbohydrate chains have been characterized in mouse, cow, and pig. In pigs, triantennary/tetraantennary neutral complex-type chains from ZPB/ZPC mixture possess stronger sperm-binding activity than those of biantennary chains (Kudo et al., 1998: Eur J Biochem 252:492-499). Most of these oligosaccharides have beta-galactosyl residues at the nonreducing ends. This study used two in vitro competition assays to investigate the participation of the nonreducing terminal beta-galactosyl residues of the ligand active chains in porcine sperm binding. The removal of the nonreducing terminal beta-galactosyl residues from either the ligand active carbohydrate chains or endo-beta-galactosidase-digested glycoproteins significantly reduced their inhibition of sperm-egg binding, indicating that the beta-galactosyl residues at the nonreducing ends are involved in porcine sperm-egg binding. A correlation between the sperm-binding activity and in vitro fertilization rate is also presented.

A detailed deletion map of chromosome 20 in human oral squamous cell carcinoma.

Loss of heterozygosity (LOH) on the long arm of chromosome 20 (20q) has been detected in several human cancers. However, little is known about LOH on chromosome 20 in oral squamous cell carcinoma (OSCC). To determine which loci of chromosome 20 were involved in OSCC tumorigenesis, 41 cases of OSCC were examined for LOH state on chromosome 20 at 17 microsatellite loci by PCR-LOH assay. LOH occurred in 41.5% of tumors in at least one locus. Among the 17 loci, D20S48 on 20p11.2 and RPN2 on 20q12-13.1 exhibited higher frequencies of LOH, 27.6% and 31.4%, respectively. The LOH incidence was significantly higher in tumors in which the primary site was on gingiva compared with other oral sites (p=0.012). Our results indicate that allelic deletions on 20q12-13.1 and 20p11.2 may play roles in OSCC carcinogenesis, and suggest that allelic deletions on 20q might have some relation with the primary site of OSCC.

Localization of a novel tumor suppressor gene loci on chromosome 9p21-22 in oral cancer.

Allelic imbalance or loss of heterozygosity (LOH) studies have been used to identify regions on chromosomes that may contain putative tumor suppressor genes. Deletions of chromosome 9 regions have been observed at high frequency in many other types of sporadic tumor, whereas in oral cancer no decisive information about the allelic loss on chromosome 9 has been reported. To provide detailed understanding of the genetic alterations in oral cancer, 24 highly polymorphic markers mapped on chromosome 9 were used to examine 34 cases of oral squamous cell carcinoma (SCC). LOH was detected in 18 (53%) of 34 informative samples at one or more loci examined. On the basis of our results, three commonly deleted regions were identified and a detailed deletion map was constructed. One of the novel regions was on 9p22, where a tumor suppressor gene, interferon a cluster (IFNA) gene, was identified before. Another region was D9S157 locus at 9p22, telomeric to IFNA locus and p15/16 genes, and the third was located on 9p21 of the D9S104 locus, centromelic to methylthioadenosine phosphorylase (MTAP) gene and p15/16 genes. Thus, our data suggest that, except for p15/16 and MTAP gene, there were at least two candidate tumor suppressor genes located at chromosome 9p, and that the alteration of these genes is associated with the tumorigenesis of oral SCC.

Localization of a novel tumor suppressor gene associated with human oral cancer on chromosome 4q25.

Recent cytogenetic and molecular studies with highly polymorphic microsatellite markers have implicated allele loss involving chromosome 4 in several human cancers, which suggests the presence of multiple tumor suppressor gene (TSG) loci. However, there has been no detailed analysis of loss of heterozygosity (LOH) on chromosome 4 in oral squamous cell carcinoma (OSCC). To determine the location of a putative TSG associated with OSCC on chromosome 4, polymerase chain reaction (PCR) analysis of microsatellite polymorphisms corresponding to 17 loci was performed to screen 32 patients with OSCC. LOH was observed in the majority of the tumors (75%) in at least one of the loci. The loci on the long arm exhibited a significantly higher frequency of deletions (66%) than those of the short arm (25%). Among the loci tested, frequent LOH was centered at D4S1573 on 4q25, which represents a region of about 4 centimorgans (cM). However, no commonly deleted regions were found on the short arm of the chromosome. We detected microsatellite instability (MI) in 31% of the cases. MI was also observed more frequently on the long arm (28%) than the short arm (6%). Thus, our data indicate that alterations of chromosome 4 regions, especially the long arm, are associated with OSCC tumorigenesis and that the 4q25 region may harbor at least one putative TSG.

Evidence of multi-step oncogenesis of oral squamous cell carcinoma.

We examined biopsy samples from one oral cancer and three precancerous lesions of the tongue of an 81-year old woman by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequence analyses using 18 oligonucleotide primer pairs of adenomatous polyposis coli (APC) gene and 5 primers of p53 gene. Normal tongue epithelium adjacent to lesions was used as a control. The four lesions harbored the common mutation of APC gene that was not detected in the control. At codon 1621 in exon 15 of the APC gene there was a C to G substitution resulting in serine (TCA) to stop codon (TGA). No mutation of p53 gene was detected in any samples of the control and three precancerous lesions of the tongue. On the other hand, an A to G substitution at codon 170 in exon 5 of p53 gene resulting in glutamic acid (ACG) to glycine (GCG) was detected in the DNA of her tongue cancer. These results may suggest that the four lesions have the same origin, and that multi-step oncogenesis had occurred, the APC gene alteration being one of the early events in the process of tumorigenesis and p53 gene alteration involved in the late events.

Allelic loss on chromosome 22 in oral cancer: possibility of the existence of a tumor suppressor gene on 22q13.

In order to understand the detail of genetic alternation on chromosome 22, we performed polymerase chain reaction analysis of microsatellite polymorphisms corresponding to 13 loci on chromosome 22. We examined 33 primary carcinoma tissues, 5 metastatic tissues and corresponding normal tissues. We detected microsatellite instability (MI) in 14 (42.4%) of 33 cases in this study. Loss of heterozygosity (LOH) was observed in at least one locus in 24 (72. 7%) of the 33 cases. Among the loci examined, LOH was restricted to D22S274 on chromosome 22q13 in 11 (40.7%) of 27 informative cases. No significant correlation between histological differentiation and LOH was observed. These observations suggest that the incidence of LOH at chromosome 22q is high and is associated with the carcinogenesis of oral squamous cell carcinoma (SCC). The D22S274 locus may play an important role in the development of oral SCC and be the site harboring a putative tumor suppressor gene.