Putative evidence for the inactivation of tumor-suppressor genes in breast carcinomas comes from reports that describe allelic losses on several chromosomes in malignant cells. However, exact genes involved have not been determined and their specific products have not been identified. Therefore, the initial goal was to identify normal breast cell products, that are lost during malignant transformation as a result of transcriptional inactivation or deletion and/or rearrangement of the corresponding genes. Using a tolerization-immunization procedure, two different glycoproteins (luminal epithelial antigen, LEA.92 and LEA.135) were identified. (a) The pattern of LEA.92 expression was detected on the apical plasma membrane of normal but not on the malignant mammary epithelial cells (MEC) in tissues. Using a culture model-system, LEA.92 was localized on non-tumorigenic immortalized MEC lines. In contrast, LEA.92 was undetectable on tumorigenic mammary carcinoma cell lines. Furthermore, binding of anti-LEA.92 antibody to non-tumorigenic MEC led to an enhanced rate of their proliferation. (b) The pattern of LEA.135 expression was found to be similar to that of LEA.92. However, unlike LEA.92, LEA.135 was also expressed on certain cases of primary breast carcinoma cells. In a retrospective study, LEA.135 expression correlated with a favorable overall survival (78% plus/minus 0.139% at >5 yrs, p=0.025) of patients with primary breast carcinomas. Finally, LEA.135 has been purified from the non-tumorigenic MEC and the first 8 amino acid of its N-terminus has been sequenced. The pattern of the antigen's expression is consistent with those of tumor-suppression genes. Through molecular cloning and functional studies, we will determine whether LEA.92 and LEA.135 are in fact products of tumor suppressor genes. Therefore, we propose to: 1) Clone and sequence full-length cDNA and their corresponding genomic clones for LEA.92 and LEA.135. These clones will be utilized in SSCP and RT-PCR to determine whether these genes have undergone mutation in breast cancer cells. The significant incidence of mutations in these genes is likely to provide evidence for their tumor-suppressor function, 2) determine whether transfected with full-length cDNA corresponding to LEA.92 and LEA.135 mRNA from normal MEC can induce a normal phenotype when expressed in tumorigenic MEC lines; conversely, determine whether transfection of non-tumorigenic MEC lines with antisense LEA.92 or LEA.135 may cause tumorigenicity, and 3) conduct a retrospective study to explore further the usefulness of LEA.135 expression as a predictive marker for good prognosis in primary breast cancer and; determine retrospectively whether LEA.92 and LEA.135 expression is associated with decrease risk of developing invasive breast carcinomas in patients initially with hyperplasia or carcinomas in situ.
| Imam, S A; Young, L; Chaiwun, B et al. (1995) Comparison of two microwave based antigen-retrieval solutions in unmasking epitopes in formalin-fixed tissue for immunostaining. Anticancer Res 15:1153-8 |
