Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5' CpG island methylation.

The p73 gene is located on 1p36.2-3, a region that is frequently deleted in human cancer. Because p73 encodes for a protein that is both structurally and functionally homologous to the p53 protein, p73 has been postulated to be a candidate tumor suppressor gene. To date, however, mutations of p73 have not been found. To study methylation of the p73 5'CpG island, a human bacterial artificial chromosome clone containing exon 1 and the 5' region of p73 was isolated. There was no evidence for p73 exon 1 methylation in normal tissues. In contrast, p73 was aberrantly methylated in approximately 30% of primary acute lymphoblastic leukemias (ALLs) and Burkitt's lymphomas. There was no evidence for methylation in any other types of hematological malignancies or solid tumors examined. In both leukemia cell lines and primary ALLs, methylation was associated with transcriptional loss of p73 by reverse transcription-PCR. We used single-strand conformational polymorphisms to screen for point mutations in a series of primary ALLs and found no mutations leading to a change in protein structure. Our results show that methylation of p73 is a frequent event in specific types of hematological malignancies and suggest that epigenetic silencing of p73 could have important consequences for cell-cycle regulation.

Deletion of p16INK4A/CDKN2 and p15INK4B in human somatic cell hybrids and hybrid-derived tumors.

Deletion or epigenetic inactivation of the tumor suppressor gene p16INK4/CDKN2 (p16) has been observed in multiple human tumors. We assayed hybrid cell lines between human diploid fibroblasts and fibrosarcoma cells for p16 allelic status and expression and found that p16 was expressed in the parental diploid fibroblast cell lines used, whereas the parental fibrosarcoma cell line HT1080.6TG exhibited homozygous deletion of p16. Most immortalized hybrid cell lines derived from these parent cell lines, whether tumorigenic or nontumorigenic, exhibited loss of fibroblast-derived p16 alleles. All p16-negative hybrid cell lines also exhibited deletion of p15INK4B (p15). Hybrid cell lines yielded tumors upon s.c. injection into athymic nude mice regardless of p16/p15 status. Tumors derived from six p16/p15-positive hybrid cells, however, revealed deletions of both p16 and p15. When human diploid fibroblasts were fused with A388.6TG squamous cell carcinoma cells, which exhibit aberrant methylation of p16, the resulting hybrids again exhibited deletion of the unmethylated fibroblast-derived p16 alleles. Transfection of both HT1080.6TG and A388.6TG cells with wild-type p16 expression vector resulted in decreased clonogenicity in culture. Although the determinants directing genetic versus epigenetic inactivation of p16 and p15 remain unclear, these results demonstrate that p16-mediated growth suppression could be abrogated by either mechanism in somatic cell hybrids.

Retention of unmethylated CpG island alleles in human diploid fibroblast x fibrosarcoma hybrids expressing high levels of DNA methyltransferase.

The mechanisms underlying ectopic methylation of CpG islands in neoplastic cells are poorly understood. One determinant may be the increased expression of DNA methyltransferase (DNA MTase) observed frequently in neoplastic cells. To evaluate the role of DNA MTase overexpression in aberrant CpG island methylation, we assessed methylation of fibroblast-derived CpG islands in human diploid fibroblast x fibrosarcoma hybrid cell lines. Each of six independently derived, immortalized hybrid cell lines exhibited a high level of DNA MTase expression, comparable to that of the fibrosarcoma parental line. The methylation status of five CpG island loci, each of which was methylated extensively in the fibrosarcoma parental cells but not in the fibroblasts, was then determined in the hybrid cell lines. The patterns of methylation were consistent and highly locus dependent among the hybrid lines. Unmethylated alleles were retained stably at three loci. The parental origin of alleles could be determined at two other loci in the hybrid cells. Whereas no methylation of parental fibroblast-derived alleles of the HIC-1 locus was noted in hybrid cell lines, a marked increase in methylation of fibroblast-derived alleles of the estrogen receptor was observed in all hybrid cell lines. Therefore, despite high-level DNA MTase expression, widespread loss of unmethylated CpG islands was not observed in the hybrid cell lines. The nonrandom pattern of increased CpG island methylation in the hybrid cell lines suggests that locus-specific features and/or clonal selection, and not just DNA MTase expression, affect the evolution of ectopic methylation in neoplastic cells. Somatic cell hybrids may provide useful models for studying aberrant epigenetic events in neoplastic cells.

p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3.

For several human tumour types, allelic loss data suggest that one or more tumour suppressor genes reside telomeric to the p53 gene at chromosome 17p13.1. In the present study we have used a new strategy, involving molecular analysis of a DNA site hypermethylated in tumour DNA, to identify a candidate gene in this region (17p13.3). Our approach has led to identification of HIC-1 (hypermethylated in cancer), a new zinc-finger transcription factor gene which is ubiquitously expressed in normal tissues, but underexpressed in different tumour cells where it is hypermethylated. Multiple characteristics of this gene, including the presence of a p53 binding site in the 5' flanking region, activation of the gene by expression of a wild-type p53 gene and suppression of G418 selectability of cultured brain, breast and colon cancer cells following insertion of the gene, make HIC-1 gene a strong candidate for a tumour suppressor gene in region 17p13.3.

Control of G1 arrest after DNA damage.

The temporal relationship between DNA damage and DNA replication may be critical in determining whether the genetic changes necessary for cellular transformation occur after DNA damage. Recent characterization of the mechanisms responsible for alterations in cell-cycle progression after DNA damage in our laboratory have implicated the p53 (tumor suppressor) protein in the G1 arrest that occurs after certain types of DNA damage. In particular, we found that levels of p53 protein increased rapidly and transiently after nonlethal doses of gamma irradiation (XRT) in hematopoietic cells with wild-type, but not mutant, p53 genes. These changes in p53 protein levels were temporally linked to a transient G1 arrest in these cells. Hematopoietic cells with mutant or absent p53 genes did not exhibit this G1 arrest, through they continued to demonstrate a G2 arrest. We recently extended these observations of a tight correlation between the status of the endogenous p53 genes and this G1 arrest after XRT and this cell-cycle alteration after XRT was then established by transfecting cells lacking endogenous p53 genes with a wild-type gene and observing acquisition of the G1 arrest and by transfecting cells processing endogenous wild-type p53 genes with a mutant p53 gene and observing loss of the G1 arrest after XRT. These observations and their significance for our understanding of the mechanisms of DNA damage-induced cellular transformation are discussed.

Expression of myeloid-associated and lymphoid-associated cell-surface antigens in acute myeloid leukemia of childhood: a Pediatric Oncology Group study.

PURPOSE: Although the expression of both myeloid- and lymphoid-associated cell-surface antigens in acute myeloid leukemia (AML) has been described, the clinical significance of such antigen expression remains unknown in the pediatric population. We sought to define an antibody panel for optimal diagnostic antigenic analysis and to test associations among antigen expression and a number of clinical features at presentation and prognosis in pediatric AML. PATIENTS AND METHODS: We reviewed the extensive immunophenotypic analysis performed at the time of diagnosis on 132 assessable patients registered on a single Pediatric Oncology Group AML protocol between 1984 and 1988. RESULTS: Eighty-eight percent of patients were identified by testing for expression of CD33 and CD13. Overall, 61% of patients expressed at least one lymphoid-associated antigen, most commonly CD4, CD7, or CD19. Expression of CD5, CD10, CD20, or CD22, commonly detected in T- or B-lineage pediatric acute lymphoid leukemia (ALL), was uncommon; coexpression of multiple lymphoid-associated antigens was also uncommon. Expression of the monocyte-associated antigen CD14 correlated with French-American-British (FAB) M4 or M5 morphology. Otherwise, no correlation between antigen expression and FAB classification was noted. None of the myeloid, lymphoid, natural-killer (NK), or progenitor-associated antigens were associated with significant differences in the likelihood of remission induction or event-free survival when expressor versus nonexpressor groups were compared. CONCLUSIONS: The distribution of cell-surface antigen expression in pediatric acute leukemia usually permitted the discrimination of AML from ALL by using a limited panel of antibodies. Although the expression of lymphoid-associated antigens was common, such expression did not seem to be associated with an adverse prognosis in pediatric AML.

Wild-type p53 is a cell cycle checkpoint determinant following irradiation.

Cell cycle checkpoints appear to contribute to an increase in cell survival and a decrease in abnormal heritable genetic changes following exposure to DNA damaging agents. Though several radiation-sensitive yeast mutants have been identified, little is known about the genes that control these responses in mammalian cells. Recent studies from our laboratory have demonstrated a close correlation between expression of wild-type p53 genes in human hematopoietic cells and their ability to arrest in G1 phase after certain types of DNA damage. In the present study, this correlation was first generalized to nonhematopoietic mammalian cells as well. A cause and effect relationship between expression of wild-type p53 and the G1 arrest that occurs after gamma irradiation was then established by demonstrating (i) acquisition of the G1 arrest after gamma irradiation following transfection of wild-type p53 genes into cells lacking endogenous p53 genes and (ii) loss of the G1 arrest after irradiation following transfection of mutant p53 genes into cells with wild-type endogenous p53 genes. A defined role for p53 (the most commonly mutated gene in human cancers) in a physiologic pathway has, to our knowledge, not been reported previously. Furthermore, these experiments illustrate one way in which a mutant p53 gene product can function in a "dominant negative" manner. Participation of p53 in this pathway suggests a mechanism for the contribution of abnormalities in p53 to tumorigenesis and genetic instability and provides a useful model for studies of the molecular mechanisms of p53 involvement in controlling the cell cycle.

Levels of p53 protein increase with maturation in human hematopoietic cells.

Transfection of the wild-type p53 gene into malignant cell lines usually results in an inhibition of proliferation. However, the physiological function of the endogenous p53 gene product has been difficult to ascertain. In order to examine whether p53 is involved in the regulation of proliferation and/or differentiation of hematopoietic tissue, we modified a recently developed flow cytometric assay to assess p53 protein expression in normal human hematopoietic cells, primary leukemias, and selected leukemia cell lines. In normal human bone marrow, p53 protein was not detected in the proliferative, progenitor cell populations identified by the cell surface antigens CD34 (progenitor cells of multiple lineages) or glycophorin (erythroid precursors). In contrast, low but detectable levels of p53 protein were observed in the nonproliferative, mature lymphoid, granulocytic, and monocytic cell populations. Similarly, p53 levels increased and DNA synthesis decreased during 12-O-tetradecanoylphorbol-13-acetate-induced differentiation of ML-1 myeloblastic leukemia cells. Both of these results suggest that endogenous, wild-type p53 protein may play a role in hematopoietic cell maturation, possibly by contributing to the inhibition of proliferation that occurs during terminal differentiation. Leukemia cells deviated from this pattern of expression: (a) in contrast to the normal, proliferative bone marrow progenitor cells, a significant percentage of patient leukemia samples expressed detectable levels of p53 protein; and (b) leukemia cell lines exhibited lineage-specific abnormalities in p53 expression, with overexpression in lymphoid cell lines and lack of expression in myeloid cell lines.