Blastic transformation of p53-deficient bone marrow cells by p210bcr/abl tyrosine kinase

Blastic transformation of chronic myelogenous leukemia (CML) is characterized by the presence of nonrandom, secondary genetic abnormalities in the majority of Philadelphia1 clones, and loss of p53 tumor suppressor gene function is a consistent finding in 25-30% of CML blast crisis patients. To test whether the functional loss of p53 plays a direct role in the transition of chronic phase to blast crisis, bone marrow cells from p53+/+ or p53-/- mice were infected with a retrovirus carrying either the wild-type BCR/ABL or the inactive kinase-deficient mutant, and were assessed for colony-forming ability. Infection of p53-/- marrow cells with wild-type BCR/ABL, but not with the kinase-deficient mutant, enhanced formation of hematopoietic colonies and induced growth factor independence at high frequency, as compared with p53+/+ marrow cells. These effects were suppressed when p53-/- marrow cells were coinfected with BCR/ ABL and wild-type p53. p53-deficient BCR/ABL-infected marrow cells had a proliferative advantage, as reflected by an increase in the fraction of S+G2 phase cells and a decrease in the number of apoptotic cells. Immunophenotyping and morphological analysis revealed that BCR/ABL-positive p53-/- cells were much less differentiated than their BCR/ABL-positive p53+/+ counterparts. Injection of immunodeficient mice with BCR/ABL-positive p53-/- cells produced a transplantable, highly aggressive, poorly differentiated acute myelogenous leukemia. In marked contrast, the disease process in mice injected with BCR/ABL-positive p53+/+ marrow cells was characterized by cell infiltrates with a more differentiated phenotype and was significantly retarded, as indicated by a much longer survival of leukemic mice. Together, these findings directly demonstrate that loss of p53 function plays an important role in blast transformation in CML.     

p53 Deficiency in liver reduces local control of survival and proliferation, but does not affect apoptosis after DNA damage

Despite good evidence for p53 dysfunction in human hepatocellular carcinomas, little is known of the significance of p53 to normal hepatocytes and whether p53 dysfunction is relevant to early hepatocarcinogenesis. We have therefore examined the consequences of targeted p53 deficiency in hepatocytes for regulation of apoptosis, proliferation, and ploidy. p53 deficiency was silent in normal liver and did not affect progression from diploidy to polyploidy in the aging liver. However, in primary culture the absence of p53 resulted in increased hepatocyte proliferation indices and decreased sensitivity to proliferation inhibition by TGFbeta. Moreover, p53-deficient cells continued to survive and proliferate under conditions of minimal trophic support that led to growth arrest and apoptosis of wild-type cells. In vivo, p53-deficient mice had enhanced proliferative responses to both xenobiotic hepatomitogen and CCl4-induced liver necrosis, although lack of persistent proliferation showed that other control mechanisms are important. There was no simple relationship between p53 and apoptosis after DNA damage because UV irradiation led to p53-independent apoptosis, even though p53 was stabilized. However, p53 did couple DNA damage to growth arrest, and abnormal mitoses after gamma-irradiation of regenerating p53 null livers demonstrated circumstances where loss of G1 and G2 checkpoints may generate abnormal ploidy. Thus p53 becomes important when hepatocytes are released from G0 and stressed, sensitizing them to mitogen and cytokine regulators of cell cycle progression and apoptosis. Hence p53 deficiency is likely to be significant in an environment of persistent regenerative stimuli and unfavorable trophic support or in the presence of other enabling genetic lesions. This model is relevant to human hepatocarcinogenesis, which almost always occurs against a background of chronic hepatocellular destruction in hepatitis and cirrhosis. In that context, by reducing the need for cytokine support and disabling DNA damage-induced growth arrest, p53 deficiency should facilitate the expansion of preneoplastic clones in chronic liver disease.     

Genetic interactions between atm and p53 influence cellular proliferation and irradiation-induced cell cycle checkpoints

Ataxia-telangiectasia and Li-Fraumeni syndrome, pleiotropic disorders caused by mutations in the genes atm and p53, share a marked increase in cancer rates. A number of studies have argued for an interaction between these two genes (for comprehensive reviews, see M. S. Meyn, Cancer Res., 55: 5991-6001, 1995, and M. F. Lavin and Y. Shiloh, Annu. Rev., Immunol., 15: 177-202, 1996). Specifically, atm is placed upstream of p53 in mediating G1-S cell cycle checkpoint control, and both atm and p53 are believed to influence cellular proliferation. To analyze the genetic interactions of atm and p53, mouse embryonic fibroblasts (MEFs) homozygously deficient for both atm and p53 were used to assess cell cycle and growth control. These double-null fibroblasts proliferate rapidly and fail to exhibit the premature growth arrest seen with atm-null MEFs. MEFs null for both atm and p53 do not express any p21(cipl/wafl), showing that p53 is required for p21(cipl/wafl) expression in an atm-null background. By contrast, homozygous loss of either atm, p53, or both results in similar abnormalities of the irradiation-induced G1-S cell cycle checkpoint. Our results suggest two separate pathways of interaction between atm and p53, one linear, involving G1-S cell cycle control, and another more complex, involving aspects of growth regulation.     

Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice

This study demonstrates that endogenously produced interferon gamma (IFN-gamma) forms the basis of a tumor surveillance system that controls development of both chemically induced and spontaneously arising tumors in mice. Compared with wild-type mice, mice lacking sensitivity to either IFN-gamma (i.e., IFN-gamma receptor-deficient mice) or all IFN family members (i.e., Stat1-deficient mice) developed tumors more rapidly and with greater frequency when challenged with different doses of the chemical carcinogen methylcholanthrene. In addition, IFN-gamma-insensitive mice developed tumors more rapidly than wild-type mice when bred onto a background deficient in the p53 tumor-suppressor gene. IFN-gamma-insensitive p53(-/-) mice also developed a broader spectrum of tumors compared with mice lacking p53 alone. Using tumor cells derived from methylcholanthrene-treated IFN-gamma-insensitive mice, we found IFN-gamma's actions to be mediated at least partly through its direct effects on the tumor cell leading to enhanced tumor cell immunogenicity. The importance and generality of this system is evidenced by the finding that certain types of human tumors become selectively unresponsive to IFN-gamma. Thus, IFN-gamma forms the basis of an extrinsic tumor-suppressor mechanism in immunocompetent hosts.     

p53-dependent DNA damage-induced apoptosis requires Fas/APO-1-independent activation of CPP32beta

In many cell types, the p53 tumor suppressor protein is required for the induction of apoptosis by DNA-damaging chemotherapy or radiation. Therefore, identification of the molecular determinants of p53-dependent cell death may aid in the design of effective therapies of p53-deficient cancers. We investigated whether p53-dependent apoptosis requires activation of CPP32beta (caspase 3), a cysteine protease that has been found to mediate apoptosis in response to ligation of the Fas molecule or to granzyme B, a component of CTL lytic granules. Irradiation-induced apoptosis was associated with p53-dependent activation of CPP32beta-related proteolysis, and normal thymocytes were protected from irradiation by Acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), a specific inhibitor of CPP32beta. We next examined whether the Fas system is required for p53-dependent apoptosis and whether stimuli that induce activation of CPP32beta induce apoptosis in p53-deficient cells. Thymocytes or activated T cells from Fas-deficient mice were resistant to apoptosis induced by ligation of Fas or CD3, respectively, but remained normally susceptible to irradiation. Thymocytes from p53-deficient mice, although resistant to DNA damage, remained sensitive to CPP32beta-mediated apoptosis induced by ligation of Fas or CD3, or by exposure to cytotoxic T cells. These results demonstrate that DNA damage-induced apoptosis of T cells requires p53-mediated activation of CPP32beta by a mechanism independent of Fas/FasL interactions and suggest that immunological or molecular methods of activating CPP32beta may be effective at inducing apoptosis in p53-deficient cancers that are resistant to conventional chemotherapy or irradiation.     

Myeloproliferative disease and myelodysplastic syndrome induced by transplantation of bone marrow cells expressing mutant p53

p53 mutations are the most common genetic alterations observed in human cancers including lymphomas and leukemias. We have previously shown that transduction of normal murine hematopoietic cells with mutant p53 alone in vitro results in an enhanced proliferative capacity and modified differentiation potential of transduced cells. In order to investigate further the role of mutant p53 in hematopoietic cell transformation, mutant p53-transduced bone marrow cells were used to reconstitute the hematopoietic system of lethally irradiated mice. The results show that overexpression of mutant p53 can initiate the transformation of immature murine hematopoietic cells in vivo and induce two types of hematopoietic disorders, myeloproliferative disease and myelodysplastic syndrome.     

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

Effect of septal perforations on measures of nasal resistance

It has been shown that p53- human colorectal cancer cells arrest after DNA damage in a G2-like state and may then undergo DNA synthesis without intervening mitosis (Waldman et al., Nature 381, 713-716, 1996). To further clarify the role of p53 in the regulation of the G2/M-phase checkpoint, we have studied cells of three closely related human lymphoblastoid cell lines (TK6, WTK1 and TK6E6, an HPV16 E6-transfected TK6 line) with differing p53 status. The cells were irradiated with 1.5-12 Gy gamma rays with or without 2 mM caffeine. There was no evidence of uncoupling of DNA synthesis and mitosis after irradiation in the p53- cell lines, WTK1 and TK6E6, suggesting that this uncoupling may not be a universal phenomenon. The apparent formation of tetraploid cells after irradiation of cells of the p53- WTK1 line was due to the occurrence of a G2-phase block in a pre-existing tetraploid population. These results support the conclusion that control of the G2/M-phase checkpoint after irradiation may differ among different cell types.     

EGFR blockade by tyrosine kinase inhibitor or monoclonal antibody inhibits growth, directs terminal differentiation and induces apoptosis in the human squamous cell carcinoma HN5

Xenotransplantation of human cells into immunodeficient mice has been used to develop models of human haemopoiesis and lymphoid cell function. However, the utility of existing mouse strains can be limited by shortened life-spans, spontaneous production of functional lymphocytes with ageing, and residual innate immunity leading to variable levels of engraftment. Mice with a deletion of the common cytokine receptor gamma chain (gamma c) gene have reduced numbers of peripheral T and B lymphocytes, and absent natural killer cell (NK) activity. A genetic cross with a recombinase activating gene 2 (RAG2)-deficient strain produced mice doubly homozygous for the gamma c and RAG2 null alleles (gamma c-/RAG2-). These mice have a stable phenotype characterized by the absence of all T lymphocyte. B lymphocyte and NK cell function. Injection of human B-lymphoblastoid cells resulted in earlier fatal metastatic lymphoproliferative disease than in NOD/LtSz-scid controls. This was particularly evident in animals injected intravenously, possibly because of residual NK activity in NOD/LtSz-scid mice. Levels of engraftment with peripheral-blood-derived human lymphocytes were also increased and associated with higher CD4/CD8 ratios. These findings demonstrate that this new strain of immunodeficient mice has significant advantages over existing strains for engraftment of human cells, and may be useful for study of adoptive immunotherapy and novel therapies for GvHD and HIV infection.     

Inactivation of isocitrate dehydrogenase kinase/phosphatase by 5''-[p-(fluorosulfonyl)benzoyl]adenosine is not due to the labeling of the invariant lysine residue found in the protein kinase family

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (-/-) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53-/- mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53-/- cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53-/- cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.     

Illegitimate recombination leading to allelic loss and unbalanced translocation in p53-mutated human lymphoblastoid cells

Allelic loss and translocation are critical mutational events in human tumorigenesis. Allelic loss, which is usually identified as loss of heterozygosity (LOH), is frequently observed at tumor suppressor loci in various kinds of human tumors. It is generally thought to result from deletion or mitotic recombination between homologous chromosomes. In this report, we demonstrate that illegitimate (nonhomologous) recombination strongly contributes to the generation of allelic loss in p53-mutated cells. Spontaneous and X-ray-induced LOH mutations at the heterozygous thymidine kinase (tk) gene, which is located on the long arm of chromosome 17, from normal (TK6) and p53-mutated (WTK-1) human lymphoblastoid cells were cytogenetically analyzed by chromosome 17 painting. We observed unbalanced translocations in 53% of LOH mutants spontaneously arising from WTK-1 cells but none spontaneously arising from TK6 cells. We postulate that illegitimate recombination was occurring between nonhomologous chromosomes after DNA replication, leading to allelic loss and unbalanced translocations in p53-mutated WTK-1 cells. X-ray irradiation, which induces DNA double-strand breaks (DSBs), enhanced the generation of unbalanced translocation more efficiently in WTK-1 than in TK6 cells. This observation implicates the wild-type p53 protein in the regulation of homologous recombination and recombinational DNA repair of DSBs and suggests a possible mechanism by which loss of p53 function may cause genomic instability.     

p53-independent apoptosis induced by paclitaxel through an indirect mechanism

The efficacy of chemotherapeutic agents may be determined by a number of different factors, including the genotype of the tumor cell. The p53 tumor suppressor gene frequently is mutated in human tumors, and this may contribute to chemotherapeutic resistance. We tested the requirement for wild-type p53 in the response of tumor cells to treatment with paclitaxel (trade name Taxol), an antineoplastic agent that stabilizes cellular microtubules. Although paclitaxel is broadly effective against human tumor xenografts in mice, including some known to carry p53 mutations, we found that p53-containing mouse tumor cells were significantly more sensitive to direct treatment with this drug than were p53-deficient tumor cells. In an attempt to reconcile this apparent discrepancy, we examined the requirement for p53 in the cytotoxic effects of tumor necrosis factor alpha (TNF-alpha), a cytokine released from murine macrophages upon paclitaxel treatment. Conditioned medium from paclitaxel-treated macrophages was capable of inducing p53-independent apoptosis when applied to transformed mouse embryonic fibroblasts and was inhibitable by antibodies against TNF-alpha. Furthermore, in response to direct treatment with TNF-alpha, both wild-type and p53-deficient tumor cells underwent apoptosis to similar extents and with similar kinetics. Our results suggest that the efficacy of paclitaxel in vivo may be due not only to its microtubule-stabilizing activity, but its ability to activate local release of an apoptosis-inducing cytokine.     

B lymphocytes are critical antigen-presenting cells for the initiation of T cell-mediated autoimmune diabetes in nonobese diabetic mice

Nonobese diabetic (NOD) mice genetically deficient in B lymphocytes (NODJg mu(null)) are resistant to T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM). Ig infusions from diabetic NOD donors did not abrogate IDDM resistance in NODJg mu(null) mice. However, T cell responses to the candidate pancreatic beta cell autoantigen glutamic acid decarboxylase (GAD), but not the control Ag keyhole limpet hemocyanin, were eliminated in NODJg mu(null) mice. To initially test whether they contribute to IDDM as APC, NOD B lymphocytes were transferred into NODJg mu(null) recipients. B lymphocytes transferred into unmanipulated NODJg mu(null) recipients were rejected by MHC class I-restricted T cells. Stable T and B lymphocyte repopulation was achieved in irradiated NODJg mu(null) mice reconstituted with syngeneic bone marrow admixed with NOD B lymphocytes. IDDM susceptibility was restored in NODJg mu(null) mice reconstituted with syngeneic marrow plus B lymphocytes, but not with syngeneic marrow only. T cell responses to GAD were restored only in NODJg mu(null) mice reconstituted with syngeneic marrow plus B lymphocytes. Hence, B lymphocytes appear to contribute to IDDM in NOD mice as APC with a preferential ability to present certain beta cell Ags such as GAD to autoreactive T cells.     

The p53-deficient mouse: a model for basic and applied cancer studies

Inactivation of the p53 gene in the germline of mice by gene targeting has provided researchers with a model similar in many respects to the analogous human inherited cancer predisposition Li-Fraumeni syndrome. The viability of p53 null mice has allowed unexpected opportunities to study the role of p53 in many different in-vivo and in-vitro contexts. Null (p53-/-) mice have an average time to tumor development of 4.5 months, while half of the heterozygous (p53+/-) mice develop tumors by 18 months. The p53-deficient mice have been particularly valuable in examining the effects of p53 loss on tumor progression. In addition, the mice hold significant promise as tools to assess carcinogens, teratogens, chemopreventative agents, and cancer therapeutic regimens.     

Role of macrophage glycosaminoglycans in the cellular catabolism of oxidized LDL by macrophages

We have previously developed an in vivo model of leukemogenesis utilizing mice reconstituted with genetically modified bone marrow cells. Based on those studies, a new single gene retroviral vector has been engineered which efficiently transfers v-myc into immature murine bone marrow cells. All reconstituted mice developed leukemia with a short latency period (5-11 weeks). In addition to hyperproliferation associated with elevated levels of PCNA, extensive apoptosis was also observed in all leukemic animals with p53 accumulating in the apoptotic cells. Whereas bax encoded protein, an effector of p53 apoptotic activity was detected in apoptotic cells, p21Waf1 protein, a potential mediator of p53 growth suppression was not detected in these cells suggesting that v-myc-induced apoptosis was independent of the ability of p53 to induce p21Waf1. These results indicate that apoptosis, a part of the cellular response to v-myc expression, does not prevent leukemia development and that hyperproliferation rather than abrogation of oncogene-induced apoptosis appears to be a critical event in v-myc-induced leukemia.     

Characterization of monoclonal antibodies directed against the alpha-subunit of the human IgE high-affinity receptor

Effective ex vivo purging techniques can decrease the likelihood of infusing bone marrow contaminated with leukemic cells during autologous transplantation. In preliminary studies, OL(1)p53, a 20-mer phosphorothioate oligonucleotide directed against p53 mRNA, decreased the number of acute myelogenous leukemia (AML) cells in vitro, suggesting a possible role for OL(1)p53 in purging bone marrow harvests of leukemia cells. To demonstrate that OL(1)p53 was nontoxic to hematopoietic progenitor cells, normal bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h, and hematopoietic progenitor cell survival was determined by in vitro colony assays. OL(1)p53 had no toxic effect on the growth of either myeloid (CFU-GM) or erythroid (BFU-E) progenitor cells. OL(1)p53 was then used to ex vivo purge bone marrow harvests from nine patients with either AML or myelodysplastic syndrome (MDS). Bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h before transplantation. The median times posttransplantation for the patient to recover an absolute neutrophil count greater than 0.5 x 10(9)/L and a platelet transfusion independence were 30 days and 56 days, respectively. Incubation of bone marrow cells with OL(1)p53 had no detrimental effect on the growth of hematopoietic progenitor cells, and transplantation of autologous bone marrow cells treated with the phosphorothioate oligonucleotide, OL(1)p53, resulted in successful recovery of circulating neutrophils following high-dose therapy in patients with AML or MDS. The data show that OL(1)p53 can be used safely to purge autologous bone marrow harvests from patients with leukemia.