Comparison ofbcr-ablProtein Expression and Philadelphia Chromosome Analyses in Chronic Myelogenous Leukemia Patients

Abstract

The Philadelphia chromosome (Ph) is found in most chronic myelogenous leukemia (CML) patients. The bcr-abl oncoprotein (P210 or PI85), the product of the fused bcr-abl gene produced by the Ph, is known to be the major factor in initiation and maintenance of the leukemic state in these types of leukemias. The authors have devised a Western blot test to detect and quantitate the bcr-abl oncoprotein in blood and bone marrow cells. The authors analyzed 1,155 peripheral blood samples from CML patients, for which there were same day Ph data, to determine if there was a correlation between bcr-abl protein levels and the percentage of Ph. A ratio of bcr-abl protein (P210 or P185) to normal abl protein (P145), the latter is ubiquitously expressed in all blood cells, has been established as a criterion for quantitating bcr-abl levels. The bcr-abl I abl protein ratio from 399 patient who were 100% Ph-positive had a mean of 2.47 (standard error |SE) of ±0.05); no false negatives were detected. A decreasing ratio was found in patients with deTrie Philadelphia chromosome is found in most patients with chronic myelogenous leukemia (CML), 15% to 25% of acute lymphocytic leukemia (ALL) and to a low extent acute myelogenous leukemia (AML). 12 The Ph is an abnormal chromosome (number 22) in which parts of chromosome 9 and 22 have been fused.

Biological effects induced by variable levels of BCR-ABL protein in the pluripotent hematopoietic cell line UT-7

“…13 However, none of these studies have determined the potentially differential effects of BCR-ABL in the same cell line according to the level of expression. The use of Western blot technology to detect and quantify BCR-ABL protein has been shown to be clinically useful in patients with CML, either at diagnosis 30 or after therapy 31 with a clear correlation between the percentage of Ph1+ metaphases. 30 However, data with regard to the anti-apoptotic effects of BCR-ABL in primary patient samples are controversial.…”

“…The use of Western blot technology to detect and quantify BCR-ABL protein has been shown to be clinically useful in patients with CML, either at diagnosis 30 or after therapy 31 with a clear correlation between the percentage of Ph1+ metaphases. 30 However, data with regard to the anti-apoptotic effects of BCR-ABL in primary patient samples are controversial. The increased resistance of primary CML progenitors to apoptosis induced by growth factor deprivation 8 was not confirmed by other studies, which found no difference between normal and CML progenitors in response to several apoptotic stimuli.…”

Bcr phosphorylated on tyrosine 177 binds Grb2

“…Brie¯y, BCR-ABL and ABL genes were inserted into the pSG5 vector (Stratagene, La Jolla, CA), which contains the early SV40 promoter to enhance in vivo expression in cells expressing the large T antigen. COS1 cells were transfected, and extracts were analysed by Western blotting with either anti-Abl (8E9) monoclonal antibody (Guo et al, 1994), anti-Bcr monoclonal antibody (7C6, Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-peptide antibodies made against Bcr 1 ± 16 (the N-terminus) or BCR(1256 ± 1271) (the C-terminus) (Campbell et al, 1990). Immunoprecipitates were denatured and fractionated by SDS gel electrophoresis, and blotted onto Immobilon ®lters for Western blotting.…”

“…Our ®ndings have shown that tyrosines 177 (Puil et al, . Blots were treated with anti-Bcr(7C6) to detect Bcr and with anti-Abl 8E9 to detect Abl, using standard ECL procedures (Guo et al, 1994) 1994), 283 and 360 (Liu et al, 1996) are phosphorylated within Bcr-Abl and Bcr. BCRN221 contained tyrosines 58, 70 and 177; BCRN159 contained tyrosines 58 and 70 of Bcr (Figure 3).…”

“…Both wild-type Bcr and Bcr Y177F were stably produced in COS1 cells (Figure 5a, lanes 2 and 3). Coexpression of P145 ABL along with Bcr reduced the level of Bcr expression by about 50% (Figure 5a, Bands were detected using ECL methods (Guo et al, 1994) To assess the degree of Grb2 binding to Bcr, antiBcr(1256 ± 1271) immunoprecipitates from the same extracts were probed with anti-Grb2 antibody ( Figure 5b). The results showed no signi®cant Grb2 binding to Bcr in untransfected COS1 cells (Figure 5b, lane 1) and only trace levels of Grb2 binding to Bcr in BCR transfected cells, either wild-type or mutant Bcr ( Figure 5b, lanes 2 and 3).…”