Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line

Abstract

Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.

The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis

“…The acute T cell leukemia cell line, CEM-CCRF, its doxorubicin (DOX)-selected and resistant P-gp high derived line CEM-A7 ϩ , and various hybrids of CCRF and A7 ϩ expressing high (IC10, 2G10) or very low (2H6 and 4G9) levels of P-gp have been previously described (21). Parental erythroblastoid leukemia K562 and K562 resistant to vincristine (VIN)-mediated death (KVIN2000) were a gift from Greg Woods (University of Tasmania, Hobart, Australia).…”

“…Multidrug-resistant and multidrug-sensitive CEM cell lines (21) were used to investigate a correlation between P-gp function and sensitivity to caspasedependent and -independent apoptosis. CEM cell lines were phenotyped for P-gp (Table 1), and P-gp high CEM cell lines were resistant to DOX-induced DNA fragmentation in comparison with P-gp low CEM cell lines (Fig.…”

Induction of MDR1 gene expression by anthracycline analogues in a human drug resistant leukaemia cell line

“…This line was derived from a classic MDR cell line CEM/A7, selected for low-level doxorubicin (DOX) resistance by stepwise selection of the parental line, CCRF-CEM, cultured in increasing concentrations of DOX (Zalcberg et al, 1994). The resistant line CEM/A7 was maintained in conditioned medium containing 0.07 µg ml -1 of DOX.…”

MRP1 not MDR1 gene expression is the predominant mechanism of acquired multidrug resistance in two prostate carcinoma cell lines

“…Two Pgp positive MDR cell lines, CEM/A7 and CEM/A7R, derived from a Pgp negative, T cell leukaemia cell line, CCRF-CEM by selection in DOX, used as positive and negative controls for the expression of MDR1. 30 Neither of the two parental lines PC-3 and DU145, nor the DOX selected PC 0.03 and DU 0.03 drug resistant sublines, expressed detectable MDR1 transcripts. In contrast, MRP1 mRNA was detected in both the parental and DOX selected drug resistant prostate lines (Figure 3).…”

“…30 Drug accumulation was performed in six-well culture plates. 1 6 10 6 cells/well of each drug resistant line and its respective parental line were plated and incubated in drug free medium overnight.…”