Primary Acute Lymphoblastic Leukemia Cells Use a Novel Promoter and 5′Noncoding Exon for the Human Reduced Folate Carrier That Encodes a Modified Carrier Translated from an Upstream Translational Start

Abstract

The human reduced folate carrier (hRFC) is reported to be regulated by up to seven alternatively spliced noncoding exons (A1, A2, A, B, C, D, and E). Noncoding exon and promoter usage was analyzed in RNAs from 27 childhood acute lymphoblastic leukemia (ALL) specimens by real-time PCR and/or 5 rapid amplification of cDNA ends (5 RACE) assay. By real-time PCR, total hRFC transcripts in ALL spanned a 289-fold range. Over 90% of hRFC transcripts were transcribed with A1, A2, and B 5 untranslated regions (UTRs). Analysis of 5 RACE clones showed that the A1 ؉ A2 5UTRs contained A1 sequence alone or a fusion of A1 and A2, implying the existence of a single, alternatively spliced 1021-bp A1/A2 noncoding region. High frequency sequence polymorphisms (AGG deletion, C/T transition) identified in the A1/A2 region by 5RACE were confirmed in normal DNAs. By reporter assays in HepG2 hepatoma and Jurkat leukemia cells, A1/A2 promoter activity was localized to a 134-bp minimal region. Translation from an upstream AUG in the A1/A2 noncoding region in-frame with the normal translation start resulted in synthesis of a larger (ϳ7 kDa) hRFC protein with transport properties altered from those for wild-type hRFC. Although there was no effect on transcript or protein stabilities, in vitro translation from A1/A2 transcripts was decreased compared with those with the B 5UTR. Our results document the importance of the hRFC A1/A2 upstream region in childhood ALL and an intricate transcriptional and posttranscriptional regulation of hRFC-A1/A2 mRNAs. Furthermore, they suggest that use of the A1/A2 5UTR may confer a transport phenotype distinct from the other 5UTRs due to altered translation efficiency and transport properties.

Prognostic Role of the Reduced Folate Carrier, the Major Membrane Transporter for Methotrexate, in Childhood Acute Lymphoblastic Leukemia: A Report from the Children's Oncology Group

“…Further, alterations in levels of hRFC were associated with clinical methotrexate resistance or decreased event-free-survival in both adult and pediatric leukemias (13,20) and in osteosarcoma patients (33). In BP-ALL lymphoblasts, hRFC transcripts are generally proportional to methotrexate uptake capacity (34) and, as further confirmed in this report, hRFC transcripts in de novo ALLs span an extraordinarily broad range (22,23,34). The latter may, in part, reflect differences in hRFC gene expression among subgroups of ALL (e.g., BP-nonhyperdiploid, BP-hyperdiploid, TEL-AML1, and E2A-PBX1; ref.…”

“…Results for hRFC were confirmed in a separate casecontrol BP-ALL population from an earlier study (21). This surprising result further documents the critical role that methotrexate plays in ALL therapy and strongly validates our previous studies of hRFC transcriptional and posttranscriptional regulation in this disease (22,23).…”

“…22), we measured relative levels of hRFC-A1/A2 and hRFC-B transcripts in this ALL cohort using primers to the hRFC-A1/A2 and hRFC-B 5 ¶ noncoding regions. Median levels for hRFC-A1/A2 and hRFC-B transcripts were 3.03 and 8.89 relative units (RU) for the 91 BP-ALL specimens, a distribution consistent with our previous findings of relative hRFC noncoding exon usage (22,23). hRFC-B transcript levels were again categorized into low and high groups using a cutoff from an AIC plot.…”

“…Recent studies on hRFC gene structure and regulation have begun to shed light on the molecular bases for variations reported in hRFC expression in primary ALL specimens (22,23,34). Transcription of hRFC in both BP-and T-cell ALL involves but two (A1/A2 and B) of the six major noncoding hRFC exons and promoters (22,23).…”

“…Transcript levels for 20 genes, for the hRFC-A1/A2 and hRFC-B noncoding exons, and 18S rRNA levels were quantitated using a LightCycler Real-time PCR machine. Reactions contained 2 AL of purified cDNA or standard plasmid, 4 mmol/L MgCl 2 , 0.5 Amol/L each of sense and antisense primers, and 2 AL FastStart DNA Master SYBR Green I enzyme-SYBR reaction mix (Roche) as described previously (22,23). Specificity of the real-time PCR amplifications was confirmed by melting curve analysis and comparisons to melting curves for standard templates.…”