Oncogene amplification has been investigated in 50 primary untreated cervical squamous carcinomas (Mitra et al., 1994). These samples originated from patients at clinical stages I and II, with histologically moderate to well differentiated carcinomas. Twenty-two proto oncogene probes were used to analyse these samples. Amplification was observed for the ERBB2 gene in 7 cases (14%) and this ranged from 5 - 6 8 copies. Two tumour samples with ERBB2 amplification showed possible mutation of the gene. The authors suggested that ERBB2 could play an important role in cervical cancer tumourigenesis.
The ERBB2 gene encodes a transmembrane protein homologous to the epidermal growth factor receptor gene mapped to 17q21 (Mitra et al, 1994). In 1991, Pinion et al. showed that ERBB2 was over-expressed in over 60% of cases of CIN III and in invasive cervical cancer . A correlation between the degree of ERBB2 amplification and histological progression was not conclusive. Mitra et al suggested in 1994 that it would be important in future to study the ERBB2 gene amplification and expression in various grades of CIN, in order to understand its role in cervical cancer evolution. They pointed out that ERBB2 amplification in CIN has not been reported before, and that Pinion and co-workers' observations had been quite encouraging for future study of CIN cases. This oncogene has also been reported to be amplified in poorly differentiated breast carcinomas (Adnane et a l , 1989).
PR ADI gene
In 1989 Arnold et a l investigated the PRAD 1 gene at l l q 13 as a proto-oncogene in parathyroid adenomas, and suggested that it was linked to the BCLl locus. The 1 lq l3 had been regarded as a region of gene amplification in various solid tumours such as oesophageal cancers (Tsuda et al, 1989), head and neck squamous carcinomas (Schuuring et a l , 1992 Berenson et al , 1989), breast (Schuuring et a l , 1992) and bladder cancers (Proctor et al, 1991). PRAD 1, HSTl, INT2 and EM Sl were identified in the same amplification unit, and the PRADl and the EMSl were shown to be over-expressed in cells
with l lq 13 amplification. (Komatsu et al., 1994 ). PRADl is also known as the Cyclin D l gene.
In 1995 Kurzrock et al reported a study of 10 cervical squamous carcinoma cell lines and 3 vulvar squamous carcinoma cell lines, using Southern and Northern blots to investigate the PRADl DNA and PRADl mRNA expression. All three vulvar cell lines showed PRADl mRNA over-expression, which was 10 fold greater than controls, and in addition two out of the three had a 5 -10 fold amplification. PRADl abnormalities were seen in 4 out of 10 squamous cervical cell lines, with over-expression of PRADl in three and a rearrangement of PRADl DNA in one. Because of the amplification and rearrangement in PRADl DNA and over-expression of PRAD mRNA, it was suggested that deregulation of PRADl, as a cell cycle regulatory gene, may be a significant molecular event in gynaecological squamous cancer evolution.
The myc Oncogene
The myc oncogene was first identified in avian myelocytomatosis viruses, retroviruses which cause acute myelocytic leukemia in chickens (Evan, 1990). The viral myc (v-myc ) gene is closely related to the cellular myc gene from a normal cell, c-myc. This c-myc gene on 8q24 is highly conserved throughout evolution. The gene has three exons and the first is non-coding. Amplification of myc has been found in colon cancer, small cell lung cancer, retinoblastoma, neuroblastoma and breast cancer among others. Cells with an amplified myc gene frequently contain double minutes (DM) or homogeneously stained regions (HSR) (1.2.7.). Increased expression of myc is frequently related to the prognosis of the tumour. An over-expression of the c-myc gene in the early stages of invasive cervical cancers has been found to be associated with a higher risk of early recurrence (Couturier et al, 1991).
N-myc and L-myc share several regions of homology with the c-myc gene and encode similar sized proteins. During embryogenesis, N-myc expression is seen at a high level in fetal brain, kidney, retina and lung. Amplification of N-myc is seen in neuroblastoma, in Wilm's tumour, in retinoblastoma and in small cell lung cancer. In neuroblastoma, the N- myc protein is expressed at a high level, but this is not caused by amplification of the gene.
The C-myc and N-myc protein elevation in the cell is also due to other mechanisms, and amplification is only one of them. Therefore, the amount of N-myc protein is a better prognostic indicator than the degree of gene amplification (Evan, 1990).
The ras oncogene
The three ras genes in mammals are Harvey- , Kirsten- and N-ras, and they are highly conserved in eukaryotes, including yeast. The first 80 aminoacids are identical in H a -, Ki- and N-ras. The second domain of 80 aminoacids are less conserved, and the third region is hypervariable. The ras genes encode 21 kDa proteins which are synthesized in the cytoplasm and attached to the inner surface of the plasma membrane. (Lemoine, 1990). In the past, several studies have shown that ras activation occurs in 40 % of colorectal cancers. Large villous adenomas in the colon, with malignant potential, also contain activated ras oncogenes in 40% of cases, but less than 20% of small adenomas demonstrate such a change. In adenocarcinomas of the lung, ras mutations are seen in 30% of cases.
Although ras gene mutation was not found in 30 cervical tumour samples by Bos et a l , 1988, Riou et al., 1984, found H-ras gene amplification in 8 out of 12 cervical tumour samples. It was suggested that there may be co-operation between the amplified T c l s genes
and Human Papilloma virus oncogenes. In 1988, Riou et al . investigated the c-Ha-ras-1 locus in cervical cancer, to determine its role in cervical tumour progression and its association with genetic factors. Identical c-Ha-ras-1 variable tandem repetition(VTR) alleles were detected in DNA from tumours and in matched control DNA samples. Heterozygosity for this allele was found in 28 of the 42 pairs tested, and the loss of one allele was found in 10 tumour samples from these 28 heterozygous patients. However, the frequency of LOH did not correlate with the stages of cervical cancer. In their study, 7 of the 10 cervical cancer samples with the c-Ha-ras-1 allele loss showed an activated c-myc gene. Similarly, all cervical tumour samples with c-Ha-ras-1 gene mutation showed an amplification or over expression of c-myc gene. Activated c-myc gene involvement in cervical cancer progression had already been shown by Riou et al, 1984 and 1985. Likewise, the c-myc gene, being a strong prognostic indicator for the recurrence of cervical tumours, was observed by
Couturier et al., 1991 (above). Accordingly, Riou et al. suggested that the c-myc and the c- Ha-ras-1 genes cooperate in the progression of cervical cancer. They also indicated that it would be desirable to determine mutations in other members of the ras gene family in the future , and to study them in pre-invasive lesions.