The Construction, Identification Of Breast Cancer Cell Lines And Functional Characterization Of Human Breast Cancer Susceptibility Gene BRCA1

Breast cancer is the most common malignant tumor affecting women world-wide. There are about 1.3 million women suffering from breast cancer and 0.4 million dying from it. The incidence of breast cancer ranks as the first commom malignant cancer of women in some large cities of China. Significantly, continuous breast cancer cell lines established from primary tumours contribute to studying the biologic characteristics of breast cancer and development of anti-tumor drugs. There were no reports about breast cancer cell lines derived from Chinese people. Therefore, no standard models to study the pathogenic mechanism at molecular level and cell signaling pathway of breast cancer for Asian patients. We had established 40 breast cancer cell lines from 52 breast cancer tumors by primary culture, and characterized three cell lines of them designated BC-023, BC-024 and BC-034.1. These new cell lines growing as adherent monolayer with characteristic epithelial morphology could be maintained continuously in vitro over 20 passages with the doubling time of 35,39 and 33hours, respectively.2. There were no bacteria, fungi and mycoplasma found in the three breast cancer cell lines.3. Isoenzyme analysis of lactate dehydrogenase (LD) and malate dehydrogenase (MD) demonstrated that the three cell lines were derived from human tissues and without cross-contamination of other species.4. Immunohistochemistry analysis also revealed that the three breast cancer cell lines were C-erbB-2 positive, which showed that the three newly established breast cancer cell lines are the origin of breast cancer tissues.5. The new cell lines were proved with severe tumorigenicity in vivo when injected into nude mice. Xenograft tumors were found within 10 days post-injection to the ratio of 100% and metastases were occurred occasionally (1/10) in nude mice injected with BC-024 cellsBRCA1 is a tumor-suppressor gene responsible for hereditary breast cancer. Most breast cancers are sporadic, but 5-10% are estimated to be due to inherited susceptibility. Numerous alternative splicing variants have been found since the cloning of the BRCA1 gene in 1994. Alternative splicing is an important source of genetic and proteinic diversity. In this study, the forward 1～13 exons of BRCA1 were cloned from breast cancer cell line ZR-75-30 by RT-PCR method. Sequence analysis showed that nine BRCA1 splice forms were isolated and characterized, compared with wild-type BRCA1 gene, one splice forms of which were novel. This novel splice form, BRCA1-ElaA-△2-17, deleted exon 1 lb, kept the original reading frame and accorded with GT-AG rule. The nucleotide sequence has been deposited in GenBank with accession number DQ333386. Sequence of some introns are alternative spliced and comprise a part of cDNA. The 89 nucleotides from intron 1 turn to be a part of BRCA1-E1 aA-△2-17. The 5’splice site of exon la in BRCA1-ElaA-△2-17 is the third splice site of the exon la except for the reported exon la and exon la’. BRCA1-ElaA-△2-17 was primarily expressed in A549, ACCM, ACHN, and K562 cells. Interestingly, BRCA1-ElaA-△2-17 was almost expressed in all human breast tumor tissues, but not normal breast tissues. Western blot analysis also indicated that BRCA1-ElaA-△2-17 was expressed in ZR-75-30 cell line at the protein level. There is still an ongoing debate concerning the cellular localization of BRCA1 protein in breast cancer. To investigate the localization and function of this novel BRCA1 alternative splicing variants, a GFP fusion express vector pEGFP-ElaA-△2-17 was constructed and introduced into the cultured Cos7、MCF7 and HeLa cells. GFP-ElaA-△2-17 clearly colocalized with the nuclear marker, suggesting that BRCAl-ElaA-△2-17 is primarily located in the nucleus. To further investigate the function of this novel splicing variant, MTT and FACS assays were performed in Cos7 and HeLa cells. Altogether, these results suggested that breast cancer associated gene BRCA1 may have unexpectedly a large number of splice variants.The novel splicing variant BRCA1-ElaA-△2-17 possibly plays a major role in the tumorigenesis of breast and/or ovarian cancer.