Figure 1 Representative examples of p185c-erbB-2 immunostaining in cancer cell lines. (A) BT-474 (mammary cell line with ERBB2 amplification and 3+ membrane overexpression). (B) SK-OV-3 (ovary cell line with ERBB2 amplification and 3+ membrane … Table 1 ERBB2 gene amplification, mRNA and protein levels in cancer cell lines. The p185c-erbB-2 levels were also estimated by Western blotting of whole-cell neverless extracts (Table 1 and Figure 4). As cell density has been reported to modulate p185c-erbB-2 levels in breast cancer cells (Kornilova et al, 1992), we compared the oncoprotein levels in low (50% confluence)- and high (100% confluence)-density cultures. The full-length, 185kDa protein, was detected in most analysed cells. A slight difference was observed between low- and high-density cultures of breast, ovary and pancreatic cell lines (Figure 4).
The highest p185c-erbB-2 levels were observed in BT-474 breast and SK-OV-3 ovary cancer cells (Table 1). In order to compare the protein content between the different cancer types, we attributed the value of one to the p185c-erbB-2 measured in MDA-MB-231 mammary cancer cells (Table 1). BT-474 and SK-OV-3 cells contained the highest protein levels associated with gene amplification and mRNA overexpression. Among the cells without gene amplification, HepG2 hepatocarcinoma and LNCaP prostate cancer cells were most enriched in p185c-erbB-2. All the colon cancer cell lines contained almost similar protein levels not significantly different from that of MDA-MB-231 cells. The pancreatic cell lines SU.86.
86, BxPC-3, HS766T and PANC-1 contained very low levels of p185c-erbB-2, detectable only after long exposure time. Only CF-PAC-1, Miapaca-2 and Capan-2 cells attained or slightly exceeded the MDA-MB-231 p185c-erbB-2 levels. Notice the wide variation in p185c-erbB-2 between the pancreatic cancer cells. In general, Western blotting and ICC results were in reasonably good agreement (Table 1). Figure 4 Expression of p185c-erbB-2 and erbB-2 messenger RNA in cancer cell lines. The first and the second lanes for each cell line correspond to pre-confluent and confluent cultures respectively. In order to compare the protein content in each cancer type, the … The erbB-2 mRNA levels were measured by real-time RT�CPCR. The results are summarised in Table 1 and compared to the Western blotting data.
In most cells, real-time RT�CPCR and Western blotting data were in good agreement, except for the COLO 320 cancer cells. Indeed, in these cells, the increase in transcript levels was not accompanied by an increase in protein Carfilzomib levels (Table 1). Like the protein levels, erbB-2 mRNA levels were quite high in HepG2 cells. The ERBB2 gene copy numbers were estimated by real-time PCR in all cancer cells (Table 1). The gene was not amplified in any of the prostate, colon and pancreatic cancer cells. SK-OV-3 presents a four-fold amplification of the ERBB2 gene.