Correlation between asparaginase sensitivity and asparagine synthetase protein content, but not mRNA, in acute lymphoblastic leukemia cell lines

Abstract

Background: Asparaginase (ASNase) is an essential component of most treatment protocols for childhood acute lymphoblastic leukemia (ALL). Although increased asparagine synthetase (ASNS) expression may contribute to ASNase resistance, there is conflicting data from patient samples with regard to correlation between ASNS mRNA content and ASNase sensitivity.

Procedure: Both T-cell and B-cell derived ALL cell lines were treated with ASNase and then monitored for cell proliferation, cell death, and ASNS mRNA and protein expression.

Results: Despite elevated ASNS mRNA following ASNase treatment, different ALL cell lines varied widely in translation to ASNS protein. Although ASNS mRNA levels did not consistently reflect ASNase sensitivity, there was an inverse correlation between ASNS protein and ASNase-induced cell death. Expression of ASNS in an ASNase-sensitive cell line resulted in enhanced ASNase resistance, and conversely, siRNA-mediated inhibition of ASNS expression promoted increased drug sensitivity.

Conclusions: These observations provide an explanation for the ASNase sensitivity of ALL cells and demonstrate the importance of measuring ASNS protein rather than mRNA in predicting ASNase responsiveness.

Fig. 1.

ALL cell lines show differential growth patterns and sensitivities to ASNase treatment. The five ALL-derived cell lines were diluted to 50 × 10⁴ cells/ml and treated with 1 IU/ml of ASNase for the indicated times. The cells were given fresh medium after 36 hr of treatment. Total viable cells (top panel) or percent cell viabilities (bottom panel) were plotted versus time of ASNase treatment. The results shown are the averages±standard deviations from three separate experiments.

Fig. 2.

Effect of ASNase concentration on cell death of ALL cell lines. ALL cells were diluted to 50 × 10⁴ ml⁻¹ and then a 100 μl aliquot placed in each well of a 96-well plate. After treatment with ASNase for 72 hr, the percent cell viability was determined. The results shown are the averages ± standard deviations from three separate experiments, and for clarity, data from treatments with less than 0.1 IU/ml of ASNase are shown as inserts.

Fig. 3.

ASNS mRNA and protein expression in ALL cell lines during ASNase treatment. ALL cells were diluted to 50 × 10⁴ cells/ml and treated with 1 IU/ml of ASNase for the indicated times. The medium was changed at 36 hr post-treatment. (panel A) RNA was collected at each time point and subjected to quantitative RT-PCR using primers specific for ASNS and GAPDH. ASNS mRNA levels were normalized to the GAPDH level and the fold induction relative to MOLT-4P cells at time 0 were plotted. (Panels B and C) Whole cell extracts were collected and subjected to immunoblotting for ASNS or actin (panel B) and the results were quantified by densitometry (panels B and C). In panel B, the immunoblots and the quantified data, normalized to actin levels, are shown for the basal amount of ASNS protein (time=0 hr). In panel C, the data are plotted relative to the value for MOLT4-P cells at time= 0 hr. The results shown are the averages+standard deviations from three separate experiments, and those values that are significantly different (P < 0.05) from the corresponding control are indicated with an asterisk. Panel D shows the relationship between ASNase IC₅₀ and ASNS protein or mRNA expression. For the three parental cell lines (MOLT-4P, NALM-6, and REH), the estimated IC₅₀ values for ASNase sensitivity from the data of Figure 2 were compared to the relative amount of ASNS protein (upper) or ASNS mRNA (lower) shown in panels A and C.

Fig. 4.

ASNS protein content in childhood ALL primary patient samples. Whole cell extracts were prepared from ALL patient samples obtained at the time of initial diagnosis. The samples (15 μg/lane) were analyzed by immunoblotting for either ASNS or actin protein levels. As a reference for relative abundance, cell extract from MOLT-4P and MOLT-4R cells was also included.