Frequent inactivation of the p73 gene by abnormal methylation or LOH in non-Hodgkin's lymphomas

p73 is a candidate tumor suppressor and imprinted gene that shares significant homology with the p53 gene. It is located on 1p36, a region frequently deleted in neuroblastoma and other tumors. To investigate the pattern of inactivation of this gene in human lymphomas, we studied 59 tumors to identify abnormal methylation in exon 1 and loss of heterozygosity (LOH) at this locus. p73 was methylated in 13/50 (26%) B cell lymphomas. There was no evidence of p73 methylation in the 9 T cell lymphomas analyzed. Burkitt's lymphomas showed the highest proportion of methylated cases (36%), although this alteration also affected other aggressive lymphomas such as diffuse large cell and some marginal zone lymphomas. LOH at the p73 locus was detected in 4/34 (11%) B and 1/9 (11%) T cell lymphomas. The p73 expression analysis showed absence or low level of p73 product in methylated lymphomas, whereas p73 was always detected in unmethylated tumors. We found monoallelic expression in normal peripheral blood samples, consistent with imprinting. None of the tumors showed LOH and methylation of the remaining allele simultaneously, suggesting that alteration of the expressed allele could lead to the total inactivation of the gene. Our results show that deletion or methylation of the p73 gene could be important mechanisms in suppressing p73 expression in B cell non-Hodgkin's lymphomas.     

Genetic interactions between Pten and p53 in radiation-induced lymphoma development

Genetic analysis of radiation-induced lymphomas from p53 heterozygous or null mice has revealed a high frequency of genetic alterations on mouse chromosome 19. Detailed microsatellite analysis of chromosome 19 deletions identified three independent regions of loss of heterozygosity, one of which was refined to a 0.3 Mb interval that contained the Pten tumor suppressor gene. More than 50% of radiation-induced tumors from p53+/- and p53-/- mice showed heterozygous loss of one Pten allele. In most cases, the remaining allele was wild type and expressed, suggesting that Pten is a haploinsufficient tumor suppressor gene for mouse lymphoma development. This conclusion was supported by the detection of specific intragenic deletions in Pten in tumors that retained one wild-type allele. Pten heterozygous mice were just as sensitive as p53+/- mice to induction of tumors by radiation, and surprisingly, the double p53+/-Pten+/-mice were equivalent to p53 null mice in radiation sensitivity. Despite the fact that Pten appears to be a haploinsufficient tumor suppressor gene, most tumors from both the single and double heterozygous mice had lost the remaining wild-type allele. The mechanism of loss in all cases involved the complete chromosome, suggesting that it is driven by other tumor suppressor genes on this chromosome. This sensitized screen therefore identified complementary roles for Pten and p53 pathways in suppression of tumor development induced by radiation exposure.     

Increased tumor cell proliferation in murine tumors with decreasing dosage of wild-type p53

A number of transgenic animal model systems have addressed the mechanistic role of p53 loss in tumor progression. However, many of these tumor models have analyzed p53 function in the context of other transgenes expressing activated oncogenes or defective tumor suppressor genes generated by gene targeting. To examine the role of p53 loss independent of other exogenous oncogenic influences, we analyzed some of the biological aspects of tumor formation and progression in p53-knockout mice containing a null germline p53 allele. We analyzed tumors from p53-/-, p53+/-, and p53+/+ littermates. Some of the p53+/- tumors had lost the remaining p53 allele (p53+/- loss of heterozygosity), whereas others retained the allele (p53+/-). In this report, we show that loss or absence of p53 conferred a tumor growth advantage by increasing the rate of cellular proliferation in a p53 dosage-dependent manner. The apoptotic levels in tumor tissue were found to be modest and not significantly dependent on p53 status. These results contrast with those from some other p53-deficient tumor models, in which p53 loss was associated with more rapid tumor progression through abrogated apoptosis. Finally, as p53 has been shown to regulate certain angiogenic factors, we examined the levels of angiogenesis in p53-containing and p53-deficient tumors. We found no p53-dependent differences in the levels of tumor angiogenesis measured by intratumoral microvessel density.     

Impairment of cytoplasmic eIF6 activity restricts lymphomagenesis and tumor progression without affecting normal growth

Eukaryotic Initiation Factor 6 (eIF6) controls translation by regulating 80S subunit formation. eIF6 is overexpressed in tumors. Here, we demonstrate that eIF6 inactivation delays tumorigenesis and reduces tumor growth in?vivo. eIF6(+/-) mice resist to Myc-induced lymphomagenesis and have prolonged tumor-free survival and reduced tumor growth. eIF6(+/-) mice are also protected by p53 loss. Myc-driven lymphomas contain PKCβII and phosphorylated eIF6; eIF6 is phosphorylated by tumor-derived PKCβII, but not by the eIF4F activator mTORC1. Mutation of PKCβII phosphosite of eIF6 reduces tumor growth. Thus, eIF6 is a rate-limiting controller of initiation of translation, able to affect tumorigenesis and tumor growth. Modulation of eIF6 activity, independent from eIF4F complex, may lead to a therapeutical avenue in tumor therapy.     

Dissecting p53 tumor suppressor functions in vivo

Although the p53 tumor suppressor acts in a plethora of processes that influence cellular proliferation and survival, it remains unclear which p53 functions are essential for tumor suppression and, as a consequence, are selected against during tumor development. Using a mouse model harboring primary, genetically modified myc-driven lymphomas, we show that disruption of apoptosis downstream of p53 by Bcl2 or a dominant-negative caspase 9 confers-like p53 loss-a selective advantage, and completely alleviates pressure to inactivate p53 during lymphomagenesis. Despite their p53-null-like aggressive phenotype, apoptosis-defective lymphomas that retain intact p53 genes do not display the checkpoint defects and gross aneuploidy that are characteristic of p53 mutant tumors. Therefore, apoptosis is the only p53 function selected against during lymphoma development, whereas defective cell-cycle checkpoints and aneuploidy are mere byproducts of p53 loss.     

The p73 locus is commonly deleted in non-Hodgkin's lymphomas

Rearrangements involving the 1p36 chromosomal region occur frequently in NHL, suggesting the existence of tumor suppressor gene(s) that are important in lymphomagenesis. p73 is closely related to the tumor suppressor p53 and maps to the chromosome 1p36 region. Here we report heterozygous deletions of the p73 locus in 25% of FL and 27% of DLBCL cases, as detected by FISH. Immunohistochemical analysis showed that four out of five cases with p73 deletions also exhibited increased Ki67 expression, indicating higher proliferation rates of the tumor cells. Our results demonstrate a high proportion of p73 locus specific deletions in NHL and suggest that deletion of this locus may play a role in the progression of NHL.     

Loss of CBP causes T cell lymphomagenesis in synergy with p27Kip1 insufficiency

CBP can function as a tumor suppressor, but the mechanisms that govern oncogenesis in its absence are unknown. Here we show that CBP inactivation in mouse thymocytes leads to lymphoma. Although CBP has been implicated in the transactivation functions of p53, development of these tumors does not seem to involve loss of p53 activity. CBP-null tumors show reduced levels of p27Kip1 and increased levels of cyclin E and Skp2, two oncoproteins that can promote p27Kip1 proteolysis. Reduction of p27Kip1 by introduction of a p27Kip1-null allele into CBP knockout mice accelerates lymphomagenesis and seems to obviate the requirement for Skp2 and cyclin E upregulation. These data suggest that CBP loss mediates lymphomagenesis in cooperation with a mechanism that reduces p27Kip1 abundance.     

Detailed mapping of chromosome 17p deletions reveals HIC1 as a novel tumor suppressor gene candidate telomeric to TP53 in diffuse large B-cell lymphoma

Deletions in the short arm of chromosome 17 (17p) involving the tumor suppressor TP53 occur in up to 20% of diffuse large B-cell lymphomas (DLBCLs). Although inactivation of both alleles of a tumor suppressor gene is usually required for tumor development, the overlap between TP53 deletions and mutations is poorly understood in DLBCLs, suggesting the possible existence of additional tumor suppressor genes in 17p. Using a bacterial artificial chromosome (BAC) and Phage 1 artificial chromosome (PAC) contig, we here define a minimally deleted region in DLBCLs encompassing approximately 0.8 MB telomeric to the TP53 locus. This genomic region harbors the tumor suppressor Hypermethylated in Cancer 1 (HIC1). Methylation-specific PCR demonstrated hypermethylation of HIC1 exon 1a in a substantial subset of DLBCLs, which is accompanied by simultaneous HIC1 deletion of the second allele in 90% of cases. In contrast, HIC1 inactivation by hypermethylation was rarely encountered in DLBCLs without concomitant loss of the second allele. DLBCL patients with complete inactivation of both HIC1 and TP53 may be characterized by an even inferior clinical course than patients with inactivation of TP53 alone, suggesting a functional cooperation between these two proteins. These findings strongly imply HIC1 as a novel tumor suppressor in a subset of DLBCLs.     

The role of p53 in suppression of KSHV cyclin-induced lymphomagenesis

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a cyclin D homolog, K cyclin, that is thought to promote viral oncogenesis. However, expression of K cyclin in cultured cells not only triggers cell cycle progression but also engages the p53 tumor suppressor pathway, which probably restricts the oncogenic potential of K cyclin. Therefore, to assess the tumorigenic properties of K cyclin in vivo, we transgenically targeted expression of K cyclin to the B and T lymphocyte compartments via the E micro promoter/enhancer. Around 17% of E micro -K cyclin animals develop lymphoma by 9 months of age, and all such lymphomas exhibit loss of p53. A critical role of p53 in suppressing K cyclin-induced lymphomagenesis was confirmed by the greatly accelerated onset of B and T lymphomagenesis in all E micro -K cyclin/p53(-/-) mice. However, absence of p53 did not appear to accelerate K cyclin-induced lymphomagenesis by averting apoptosis: E micro -K cyclin/p53(-/-) end-stage lymphomas contained abundant apoptotic cells, and transgenic E micro -K cyclin/p53(-/-) lymphocytes in vitro were not measurably protected from DNA damage-induced apoptosis compared with E micro -K cyclin/p53(wt) cells. Notably, whereas aneuploidy was frequently evident in pre-lymphomatous tissues, end-stage E micro -K cyclin/p53(-/-) tumors showed a near-diploid DNA content with no aberrant centrosome numbers. Nonetheless, such tumor cells did harbor more restricted genomic alterations, such as single-copy chromosome losses or gains or high-level amplifications. Together, our data support a model in which K cyclin-induced genome instability arises early in the pre-tumorigenic lymphocyte population and that loss of p53 licenses subsequent expansion of tumorigenic clones.     

Tumor predisposition in mice mutant for p63 and p73: evidence for broader tumor suppressor functions for the p53 family

p63 and p73 are functionally and structurally related to the tumor suppressor p53. However, their own role in tumor suppression is unclear. Given the p53-like properties of p63 and p73, we tested whether they are involved in tumor suppression by aging mice heterozygous for mutations in all p53 family genes and scored for spontaneous tumors. We show here that p63+/-;p73+/- mice develop spontaneous tumors. Loss of p63 and p73 can also cooperate with loss of p53 in tumor development. Mice heterozygous for mutations in both p53 and p63 or p53 and p73 displayed higher tumor burden and metastasis compared to p53+/- mice. These findings provide evidence for a broader role for the p53 family than has been previously reported.     

Loss of p53 in benzene-induced thymic lymphomas in p53+/- mice: evidence of chromosomal recombination

The purpose of this study was to examine the role of chromosomal recombination in mediating p53 loss in benzene-induced thymic lymphomas in C57BL/6-Trp53 haploinsufficient (N5) mice (p53+/- mice). We characterized loss of heterozygosity (LOH) on chromosome 11 using seven microsatellite markers in 27 benzene-induced and 6 spontaneous thymic lymphomas. Eleven patterns of LOH were found between the induced and spontaneous tumors, with only one pattern being in common between the tumor groups. Nearly 90% (24 of 27) of benzene-induced tumors exhibited loss of the functional p53 allele locus, and 83% (20 of 24) of these tumors retained two copies of the disrupted p53 allele. The results indicate that benzene induces a high frequency of LOH on chromosome 11 in p53+/- mice, likely mediated by aberrant chromosomal recombination.     

Overexpression of human O6-alkylguanine DNA alkyltransferase (AGT) prevents MNU induced lymphomas in heterozygous p53 deficient mice

O6-alkylguanine DNA alkyltransferase (AGT) is a key mechanism in the prevention against MNU induced malignant transformation by removal of O6 methyl guanine (O6mG) adducts. We asked whether heterozygous p53 deficient mice (p53+/-) would be more susceptible to MNU induced lymphomas than wild type mice, and whether O6mG adducts were responsible for this susceptibility. To determine whether MGMT overexpression would be protective, p53+/- mice were bred to human MGMT transgenic mice (MGMT+) and treated with 50 mg/kg MNU. MNU increased the incidence of thymic lymphomas in non-transgenic p53+/- mice from 23% (n=13) to 68% (n=22) and decreased the mean latency from 433 to 106 days (P=0.01 compared to untreated mice). Wild type mice had an incidence of 30% (n=38) and a mean latency of 135 days after MNU. Overexpression of MGMT in the thymus of p53+/- mice significantly reduced the lymphoma incidence from 68 to 28% (n=17) and increased the latency from 106 to 167 days (P=0.003). Similarly, the lymphoma incidence in MGMT+/wild type mice decreased from 30 to 8% (n=12) and the latency increased to 297 days (P=0.2). Loss of the wild type allele was found in only 2/17 lymphomas occurring in p53+/- mice and there were no significant point mutations in exons 5-8 of p53. Furthermore, there was no loss of p53 function in these mice. These data demonstrate that unrepaired O6mG lesions act cooperatively with the reduced p53 dose and lead to lymphomagenesis in p53+/- mice, but AGT overexpression and rapid removal of O6mG adducts is protective.     

Suppression of thymic lymphomas and increased nonthymic lymphomagenesis in Trp53-deficient mice lacking inducible nitric oxide synthase gene

Trp53-deficient mice spontaneously develop lymphomas, mainly of thymic origin, although the molecular mechanism remains largely unknown. As several interaction effects between p53 and iNOS have been reported, we hypothesized that iNOS activity in the thymus is causally linked to lymphomagenesis in Trp53-deficient mice. We therefore created mouse strains with different combinations of the Trp53 and iNOS genes. Western blot and histologic analyses showed that the iNOS protein was constitutively expressed in the thymus independently of Trp53 status and its expression was enhanced in Trp53+/- and Trp53-/- mice compared to Trp53+/+ mice. Homozygous disruption of iNOS decreased the incidence of thymic lymphomas by almost 40% (p=0.087) and 90% (p<0.05) in Trp53-/- and Trp53+/- mice, respectively, compared to the respective iNOS wild-type mice but significantly (p<0.05) increased the development of nonthymic lymphomas in Trp53-/- and Trp53+/- mice. Although iNOS gene disruption did not affect the phenotype of thymic lymphomas, absence of the iNOS gene shifted the spectrum of nonthymic lymphoma from the B-cell to the T-cell lineage. RT-PCR analysis revealed enhanced expression of IL-10, which could have a promoting effect on lymphomagenesis, even without any stimulation, in the spleen of aging mice with the gene combinations Trp53-/-iNOS-/- and Trp53+/-iNOS-/- but not Trp53-/-iNOS+/+ or Trp53+/-iNOS+/+. These results suggest that iNOS could increase the development of thymic lymphomas in Trp53-deficient mice. While iNOS may have protective effects against nonthymic lymphomagenesis, the regulation of cytokine production by iNOS may be involved in the underlying mechanism of antilymphomagenesis effects in the peripheral lymphoid organ.     

Hipk2 cooperates with p53 to suppress γ-ray radiation-induced mouse thymic lymphoma

A genome-wide screen for genetic alterations in radiation-induced thymic lymphomas generated from p53+/- and p53-/- mice showed frequent loss of heterozygosity (LOH) on chromosome 6. Fine mapping of these LOH regions revealed three non-overlapping regions, one of which was refined to a 0.2 Mb interval that contained only the gene encoding homeobox-interacting protein kinase 2 (Hipk2). More than 30% of radiation-induced tumors from both p53+/- and p53-/- mice showed heterozygous loss of one Hipk2 allele. Mice carrying a single inactive allele of Hipk2 in the germline were susceptible to induction of tumors by γ-radiation, but most tumors retained and expressed the wild-type allele, suggesting that Hipk2 is a haploinsufficient tumor suppressor gene for mouse lymphoma development. Heterozygous loss of both Hipk2 and p53 confers strong sensitization to radiation-induced lymphoma. We conclude that Hipk2 is a haploinsufficient lymphoma suppressor gene.     

Loss of p53 in F-MuLV induced-erythroleukemias accelerates the acquisition of mutational events that confers immortality and growth factor independence.

Erythroleukemias induced by Friend Murine Leukemia Virus (F-MuLV) involve the insertional activation of the proto-oncogene Fli-1, and the inactivation of the p53 tumor suppressor gene. While the activation of Fli-1 is an early, primary transforming event, p53 mutations are correlated with the immortalization of erythroleukemic cells in culture. In this study we have further analysed the role of p53 loss in F-MuLV induced erythroleukemias by examining the progression of this disease in p53 deficient mice. We found that p53-/- mice succumb to the disease more rapidly than p53+/+ littermates. Additionally, of the 112 tumors generated, 19 gave rise to immortal cell lines, eight of which were derived from p53-/- mice, and ten of which were from p53+/- mice. The ability of these primary tumor cells to grow in culture was associated with the complete loss of wild-type p53 in these cell lines. However, cells from many of the tumors induced in p53-/- hosts did not survive in vitro. These results suggest that the loss of p53 does not directly immortalize tumor cells. Instead, we have evidence to suggest that the loss of p53 promotes the accumulation of mutations that are required for survival in culture and that are capable of accelerating tumor progression in vivo. Indeed, mutations causing expression of the growth factor gene erythropoietin (Epo), were detected in two of seven Epo-independent cell lines from p53 deficient primary erythroleukemias. Moreover, the mechanism of activation of the Epo gene in one of these two Epo-independent cell lines involved genomic rearrangement, that is a hallmark of genetic instability. We propose that, in F-MuLV induced-erythroleukemias, p53 loss may encourage the accumulation of further mutations, subsequently conferring a growth advantage and immortality to the transformed erythroblasts.     

Targeting p73 in cancer

p73 is a member of the p53 family of tumor suppressors. Transactivating isoforms of p73 (TAp73) have p53-like, anti-proliferative and pro-apoptotic activities that are crucial for an efficient chemotherapy response. In line with this, genetic studies in mice have confirmed that TAp73 acts as a tumor suppressor. However, in contrast to p53, which is commonly inactivated in human cancer by point mutations, the TP73 gene is almost never mutated. Instead, the tumor suppressor activity of TAp73 is inhibited through a variety of mechanisms including epigenetic silencing and complex formation with inhibitory proteins. All these mechanisms have in common that they are in principle reversible and therefore amenable to therapeutic intervention. Here, we will review how tumor cells control the tumor suppressor activity of TAp73 and discuss possible strategies targeting p73 for reactivation.     

p63 and p73 do not contribute to p53-mediated lymphoma suppressor activity in vivo

p53 is one of the most important tumor suppressor genes in human cancer, but the roles of its homologues p63 and p73 in tumor suppression, alone or in collaboration with p53, remains controversial. Both p63 and p73 can be deregulated after DNA damage, and induce cell cycle arrest and apoptosis, but mice carrying inactive alleles of these genes do not develop spontaneous tumors. Since heterozygous loss of p53 confers strong sensitization to radiation-induced lymphoma development, we investigated the possibility that radiation exposure may reveal previously undetected tumor suppressor properties in p63 or p73, alone or in combination with p53. Animals heterozygous for p63 or p73, as well as both double heterozygous p53/p63 or p53/p73 mice, showed no significant differences in tumor latency, spectrum or frequency after gamma-radiation, compared to their control counterparts. Deletions were found near the p63 locus on chromosome 16 in radiation-induced tumors, but these frequently included the knockout allele. No deletions or LOH involving the p73 gene were detected, and expression of both genes was maintained in the tumors. We conclude that p53 homologues do not contribute to p53 tumor suppressor activity in lymphoma development.     

The role of p73 in hematological malignancies.

The P73 gene is a homologue of the P53 tumor suppressor. Owing to its structural similarity with p53, p73 was originally considered to have tumor suppressor function. However, the discovery of N-terminal truncated isoforms with oncogenic properties showed a 'two in one' structure of its product, p73 protein. The full-length variants are strong inducers of apoptosis, whereas the truncated isoforms inhibit proapoptotic activity of p53 and the full-length p73. Thus, p73 is involved in the regulation of cell cycle, cell death and development. Moreover, it plays a role in carcinogenesis and controls tumor sensitivity to treatment. p73 is commonly expressed in tumor cells in hematological malignancies. Overexpression of p73 protein and aberrant expression of its particular isoforms, with very low frequency of P73 hypermethylation or mutations, were found in malignant myeloproliferations, including acute myeloblastic leukemia. In contrast, hypermethylation and subsequent inactivation of the P73 gene are the most common findings in malignant lymphoproliferative disorders, especially acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphomas. Assessment of P73 methylation may provide important prognostic information, as was confirmed in patients with ALL. This review summarizes some aspects of p73 biology with particular reference to its possible pathogenetic role and prognostic significance in hematological malignancies.