John C. Law

Mutational inactivation of the p53 gene in the human erythroid leukemic K562 cell line

The K562 human chronic myelogenous leukemia (CML) cell line has attained widespread use as a model for studying hematologic malignancy and erythroid differentiation. Sequencing of the p53 gene in the K562 cell line demonstrated a mutation in exon 5 characterized by a single base insertion (cytosine) between codons 135 and 136. This frameshift mutation leads to an N-terminal truncated protein of 147 amino acids. Only the mutated sequence was present suggesting that the normal allele has been lost. Reverse transcription PCR (RT-PCR) detected a p53 transcript but Western blotting and immunohistochemical staining of cells failed to detect p53 protein. The identification of an inactivation mutation of p53 in the K562 cell line further supports the argument that p53 mutations play a role in myeloid blast transformation of CML.

Association between human cancer and two polymorphisms occurring together in the p21Waf1/Cip1 cyclin-dependent kinase inhibitor gene†

The cyclin‐dependent kinase inhibitor gene p21Waf1/Cip1 plays a role in signaling cellular growth arrest. In response to DNA damage, p21 is induced by the p53 gene, thereby playing a direct role in mediating p53‐induced G1 arrest. Alterations in this gene may adversely affect regulation of cellular proliferation and increase susceptibility for cancer. Two polymorphisms have previously been characterized in the p21 gene: a C→A transversion at codon 31 (ser→arg) and a C→T transition 20 nucleotides downstream from the 3 end of exon 3.

Loss of heterozygosity of the short arm of chromosomes 3 and 9 in oral cancer

Loss of heterozygosity (LOH) on chromosomes 3p and 9p has been documented in a variety of malignancies, which suggests the presence of tumor suppressor gene loci on these chromosomes. We have studied 77 oral carcinomas for LOH using 16 microsatellite markers distributed over 5 human chromosomes. Fifty‐five (71%) of these tumors showed LOH at one or more loci. A significant proportion of LOH at the informative tumors was observed at chromosomes 3p and 9p: 58% and 48%, respectively. A majority of the tumors showed losses at multiple loci on chromosomes 3p or 9p or on both. Our results suggest that tumor suppressor genes located on the short arms of chromosomes 3 and 9 may be involved in the pathogenesis of oral carcinoma. These regions of deletion observed in oral cancers overlap those reported in other neoplasms. However, we did not find any evidence of these changes in tumor margins with early pathological changes.

Molecular analysis of the IL-2 receptor β chain gene expressed in human tumor cells

Interleukin-2 (IL-2) is recognized as a T cell growth factor. We have previously reported that human carcinoma cell lines are inhibited in growth by exogenous IL-2, which binds to the IL-2 receptor β (IL-2Rβ) chain ubiquitously expressed on the surface of tumor cells. A possibility was considered that IL-2Rβ on carcinomas responsible for negative signaling was different from that expressed on hematopoietic cells. To investigate this possibility, mRNA for the IL-2Rβ chain was amplified and compared in carcinoma and lymphoid cells. Using RT–PCR with pairs of sense-antisense oligonucleotide primers specific for the various regions of extracellular, transmembrane and intracellular domains of the IL-2Rβ chain, we amplified mRNA obtained from three human carcinoma cell lines and human lymphoid cells as controls. The identity of the amplicons was confirmed by Southern analysis with the 32P-labeled cDNA probe coding for the entire span of the IL-2Rβ chain. In addition, genomic DNA obtained from the tumor cell lines was sequenced to examine the possibility that a mutation is present in the gene coding for the intracellular IL-2Rβ chain domain. No mutations or deletions were detected. The message for all three domains of the β chain was identical in tumor cells and in normal lymphoid cells used as controls. Also, by Western blot and northern analyses no differences between IL-2Rβ chain in tumors vs that expressed in lymphoid cells were demonstrable. The IL-2Rγ chain, which participates in IL-2/IL-2R signaling pathway, was expressed in tumor cells. Expression of JAK1 transcripts in these cells was comparable to that in lymphocytes. However, RT–PCR analysis identified differences in expression of JAK3 splice variants (B and M) in tumor cells. These differences may be responsible for altered downstream signaling by IL-2. Overall, our data indicate that the same IL-2/IL-2R pathway is operative in human carcinomas and in normal epithelial or lymphoid cells.

Identification of a PstI polymorphism in the p21Cip1/Waf1 cyclin-dependent kinase inhibitor gene.

A PstI polymorphism in the 3′ flanking region of the p21CiP1/Waf1 cyclin-dependent kinase inhibitor gene is described. DNA sequencing analysis identified a C→T base substitution in the 3′ flanking region of the gene. This substitution leads to the destruction of a PstI site and results in a biallelic DNA polymorphism. This restriction fragment length polymorphism (RFLP) provides the first known genetic marker for this cell cycle regulatory gene.