Coexistence of multiple mechanisms of PT523 resistance in human leukemia cells harboring 3 reduced folate carrier alleles: transcriptional silencing, inactivating mutations, and allele loss by Yotam Kaufman, Ilan Ifergan, Lilah Rothem, Gerrit Jansen, and Yehuda G. Assaraf Blood Volume 107(8):3288-3294 April 15, 2006 ©2006 by American Society of Hematology

Antifolate growth inhibition of parental human leukemia CCRF-CEM cells and their various PT523-resistant sublines. Antifolate growth inhibition of parental human leukemia CCRF-CEM cells and their various PT523-resistant sublines. Exponentially growing parental CCRF-CEM (WT, ○) cells and their various PT523-resistant sublines PTR0.03 (•), PTR0.05 (▪), and PTR0.5 (▴) were exposed to increasing concentrations of GW1843 (A) or pemetrexed (MTA) (B) as detailed in “Materials and methods.” Following 3 days of incubation at 37°C, viable cell counting was performed using trypan blue exclusion. Values depicted represent means ± SD of 3 to 5 independent experiments. Yotam Kaufman et al. Blood 2006;107:3288-3294 ©2006 by American Society of Hematology

Initial rates of [3H]MTX uptake in CCRF-CEM cells and their various PT523-resistant sublines. Initial rates of [3H]MTX uptake in CCRF-CEM cells and their various PT523-resistant sublines. Exponentially growing cells were washed, resuspended in a HEPES-buffered saline solution at pH 7.4 containing 2 μM [3H]MTX, and incubated at 37°C for 3 minutes. Cells were then washed 3 times with ice-cold buffer, centrifuged, lysed, and processed for scintillation counting as described in “Materials and methods.” Influx rates are given as pmol [3H]MTX/min/107 cells and are presented as the means ± SD of 3 to 6 independent experiments performed on different days. The influx rate of [3H]MTX in parental (WT) cells was 3.9 ± 0.64 pmol [3H]MTX/min/107 cells. The influx rate of WT cells was referred to as 100% and the residual percentage of [3H]MTX transport in the various sublines relative to parental cells is also depicted. Yotam Kaufman et al. Blood 2006;107:3288-3294 ©2006 by American Society of Hematology

Genomic PCR-SSCP analysis of RFC exon 3 in parental cells and their PT523-resistant sublines. Genomic PCR-SSCP analysis of RFC exon 3 in parental cells and their PT523-resistant sublines. High-molecular-weight gDNA (∼50 ng) isolated from parental CCRF-CEM cells and their various antifolate-resistant sublines was PCR amplified in the presence of [32P]dCTP using oligonucleotide primers targeting exon 3. The PCR products were resolved by electrophoresis on nondenaturing, 8% polyacrylamide gels, as described in “Materials and methods.” The arrowhead shown on the right side depicts the band with the altered electrophoretic mobility. Yotam Kaufman et al. Blood 2006;107:3288-3294 ©2006 by American Society of Hematology

Northern blot analysis of RFC mRNA levels in parental CCRF-CEM cells and their PT523-resistant sublines. Northern blot analysis of RFC mRNA levels in parental CCRF-CEM cells and their PT523-resistant sublines. Total RNA (30 μg, except for RFC overexpressing CEM-7A cells, which contained only 6 μg) from WT cells and the various PT523-resistant sublines was fractionated by electrophoresis on 1.2% agarose gels containing formaldehyde, blotted onto Zeta-Probe-GT nylon membrane (Bio-Rad) and probed with a [32P]oligolabeled full-length RFC cDNA13 (A). The blots were then washed under high stringency conditions and exposed to x-ray films. The intensity of the 3-kb RFC transcript was determined by scanning densitometry and normalized to the methylene blue staining of the 18S and 28S rRNA bands (B). The ratio of the intensity of the RFC transcript in each cell line versus WT cells is depicted in the bottom of panel A. Yotam Kaufman et al. Blood 2006;107:3288-3294 ©2006 by American Society of Hematology

Western blot analysis of RFC expression in CCRF-CEM cells and their PT523-resistant sublines. Western blot analysis of RFC expression in CCRF-CEM cells and their PT523-resistant sublines. Microsomes were isolated from exponentially growing cells and proteins were extracted using a Triton X-100-containing buffer as detailed in “Materials and methods.” Aliquots of microsomal proteins (75 μg) from RFC overxepressing CEM-7A cells, WT cells, and their PT523-resistant sublines were resolved by electrophoresis on 10% polyacrylamide gels containing SDS and electroblotted onto a Nytran nylon membrane (Schleicher and Schuell). Then, the blots were reacted with a peptide antiserum against a C-terminal peptide of human RFC9 and detected by enhanced chemiluminescence (A). An internal 148-kDa protein detected during the Panceau S staining was used to normalize for any differences in protein loading (B). Yotam Kaufman et al. Blood 2006;107:3288-3294 ©2006 by American Society of Hematology

Southern blot analysis of RFC gene copy number in parental WT cells and their PT523-resistant variants. Southern blot analysis of RFC gene copy number in parental WT cells and their PT523-resistant variants. High-molecular-weight gDNA (15 μg) was codigested with MfeI and XmaI (a 6-cutter; A) or MfeI and MspI (a 4-cutter; C), fractionated by electrophoresis on 0.8% agarose gels, and transferred to Zeta-Probe-GT nylon membranes. The blots were then hybridized with a [32P]labeled 5′-genomic hRFC probe as detailed in “Materials and methods.” Following hybridization at 65°C, the membranes were washed under high stringency conditions and exposed to x-ray film. The intensity of the RFC band in panels A and C was estimated by scanning densitometry and normalization to an approximate 1.5-kb repetitive DNA band observed after ethidium bromide staining (B). The band obtained in panel A and the middle band observed in panel C (arrowhead) are both approximately 600 bp in length. The arrows shown on the right side in panel C denote the WT bands that were lost in the PT523-resistant cells. Yotam Kaufman et al. Blood 2006;107:3288-3294 ©2006 by American Society of Hematology