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

Zalcberg, John; Hu, Xiangdong; Wall, Dominic; Mirski, Shelagh E. L.; Cole, Susan P.C.; Nadalin, Gabriella; Luise, M. De; Parkin, John; Vrazas, Vickie; Campbell, Lynda J.; Kantharidis, Phillip

doi:10.1002/ijc.2910570414

Cited by 32 publications

(22 citation statements)

References 12 publications

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“…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).…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: P-gp Confersmentioning

confidence: 99%

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

Smyth¹,

Krasovskis²,

Sutton³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

335

231

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Multidrug resistance mediated by the drug eff lux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspasedependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inf licted by these stimuli were defined as caspase dependent by various soluble peptide f luoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to eff luxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.

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Section: Methodsmentioning

confidence: 99%

Section: P-gp Confersmentioning

confidence: 99%

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

Smyth¹,

Krasovskis²,

Sutton³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

335

231

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Cell Lines and Drug Treatmentmentioning

confidence: 99%

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

Slater

Rischin

et al. 1999

Br J Cancer

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SummaryThe effects of 4-demethoxydaunorubicin (idarubicin, IDA) and MX2, a new morpholino-anthracycline, on up-regulation of the MDR1 gene in the low-level multidrug resistant (MDR) cell line CEM/A7R were compared at similar concentrations (IC 10 , IC 50 and IC 90 ) over a short time exposure (4 and 24 h). The chemosensitivity of each drug was determined by a 3-day cell growth inhibition assay. Compared with epirubicin (EPI), IDA and MX2 were 17-and eightfold more effective in the CEM/A7R line respectively. No cross-resistance to 5-FU was seen in the CEM/A7R line. Verapamil (5 µM) and PSC 833 (1 µM), which dramatically reversed resistance to EPI in the CEM/A7R line, had no sensitizing effect on the resistance of this line to MX2, but slightly decreased resistance to IDA. The sensitivity to 5-FU was unchanged by these modulators. The induction of MDR1 mRNA expression by IDA, MX2 and 5-FU was analysed by Northern blotting and semiquantitatively assessed by scanning Northern blots on a phosphorimager. The relative level of MDR1 expression was expressed as a ratio of MDR1 mRNA to the internal RNA control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). IDA, MX2 and 5-FU differentially up-regulated MDR1 mRNA in the CEM/A7R line in a dose-dependent manner. Both IDA and MX2 induced MDR1 expression within 4 h. 5-FU up-regulated MDR1 expression only when drug exposure was prolonged to 24 h. Based on MRK 16 binding, flow cytometric analysis of P-glycoprotein (Pgp) expression paralleled the increase in MDR1 mRNA levels. For the three anthracyclines, the increase in MDR1 expression was stable in cells grown in the absence of drug for more than 3 weeks after drug treatment. The induction of MDR1 expression by 5-FU was transient, associated with a rapid decrease in the increased Pgp levels which returned to baseline 72 h after the removal of 5-FU. This study demonstrates that MDR1 expression can be induced by analogues of anthracyclies not pumped by Pgp, and that this induction appears to be stable despite a 3-week drug-free period.

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“…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).…”

Section: Northern Analysis Of Mrp1 and Mdr1 Mrnamentioning

confidence: 96%

“…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.…”

Section: Drug Accumulationmentioning

confidence: 99%

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

Zalcberg

Slater

et al. 2000

Prostate Cancer Prostatic Dis

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Multidrug resistant prostate cancer cell lines DU 0.03 and PC 0.03 were established from the parental prostate cancer cell lines DU145 and PC-3 respectively by stepwise selection in doxorubicin (DOX) from 0.001 to 0.03 m mg/ml. As cells adapted to each concentration of DOX. the drug concentration was increased by 0.001 m mg/ml. The chemosensitivity of each line was determined by growth inhibition assay. The DU 0.03 and PC 0.03 lines exhibit a 5 ± 10-fold and 1.3 ± 2.8-fold increase in resistance to anthracyclines, vinblastine (VLB) and mitozantrone (Mito), respectively. Verapamil (5 m mM) partially reversed the resistance to the anthracycline and completely reversed the resistance to VLB and Mito. Drug kinetic studies measured by intracellular accumulation of 3 H-daunorubicin demonstrated a 3 fold decrease in the level of intracellular 3 H-daunorubicin in the PC 0.03 and DU 0.03 resistant lines compared with their respective parental line. This effect was partially reversed by 5 m mM verapamil. The expression of MDR1 and MRP genes was analysed by Northern blotting and RT-PCR. P-glycoprotein (Pgp) and MRP protein were tested by immunocytochemistry staining using the monoclonal antibodies J-SB1. C219 and MRK16 (Pgp) and MRPm6 and MRPr1 (MRP). Neither Northern blot analysis nor the more sensitive RT-PCR demonstrated detectable MDR1 transcripts in any of the prostate cancer cell lines and the three Pgp monoclonal antibodies failed to reveal expression of Pgp.A 2 ± 4-fold increase in MRP1 mRNA levels in the drug resistant DU 0.03 and PC 0.03 lines were demonstrated by both Northern blotting and RT-PCR consistent with the ®ndings observed after staining by the two speci®c monoclonal antibodies, MRPm6 and MRPr1. Southern blot analysis demonstrated a 2-fold increase in the MRP1 gene copy number in the PC 0.03 line but not in the DU 0.03 line, suggesting that the overexpression of the MRP gene was regulated at the level of transcription in the latter line. We conclude that MRP1 not MDR1 overexpression. contributes to acquired drug resistance in these two prostate cancer cell lines. Prostate Cancer and Prostatic Diseases (2000) 3, 66±75.

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

Cited by 32 publications

References 12 publications

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

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

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

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

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