ALTERNATIVE SPLICING OF THE P53 TUMOR-SUPPRESSOR GENE IN THE MOLT-4 T-LYMPHOBLASTIC LEUKEMIA-CELL LINE

The expression of the p53 tumor suppressor gene in ten human cell line s (nine cancers and one normal) was studied using reverse transcriptio n, polymerase chain reaction (PCR) and direct sequencing. Using P53U a nd P53D primers for amplifying a 371-base pair (bp) target fragment sp anning exons 7-10 of p53 cDNA, normal-sized PCR products were amplifie d from 9 cell lines but not from the Hep3B hepatocellular carcinoma (H CC) cell line. An additional larger band (504 bp) was observed for the Molt-4 T-lymphoblastic leukemia cell line. Employing P531 and P53D pr imers which flank a 76-bp p53 cDNA fragment, 76 bp as well as 209 bp p roducts were generated by PCR of Molt-4 cDNA. Direct sequencing of the 504 bp and 209 bp bands confirmed the presence of a 133 bp insertion between exons 9 and 10 in the aberrant transcript. This insertion was homologous to a 130-bp sequence within the wild-type p53 intron 9, exc ept for 2 point mutations and 3 base insertions. Sequencing of P53U/P5 3D PCR products of Molt-4 genomic DNA revealed an 8 bp deletion just d ownstream to the 133 bp insertion, creating a novel donor splicing sit e within intron 9. This site, coupled with an inherent acceptor splici ng site just upstream to the 133 bp insertion, suggests that the 133 b p stretch represents an alternative exon. The occurrence of a terminat ion signal within this alternative transcript is predicted to culminat e in a truncated p53 translational product. The sequences of the 371 b p PCR products of Molt-4, HT-1080, SiHa, CaSki, HeLa and MRC-5 cell li nes corresponded with the wild-type p53 cDNA. G --> T transversions at the third base of codon 249 of p53 were detected in Mahlavu and PLC/P RF/5 HCC lines, while a TAC to CAC mutation at codon 234 was observed in an allele of the Raji Burkitt lymphoma line.