P53-negative Cells Research Articles

Phorbol ester stimulated Cip1 expression in p53-negative leukemic cells

In differentiating leukemic cells, cyclin-dependent kinase interacting protein (Cip1) is induced and stimulates a G(1) arrest. TPA treated U937 monoblastoid cells expressed Cip1, hypophosphorylated retinoblastoma protein (Rb), arrested in G(1) and differentiated. PKC-zeta cells are U937 cells that overexpress the zeta isoform and display alterations in endogenous PKC isoform expression. TPA treated PKC-zeta cells undergo apoptosis without differentiating. TPA treated PKC-zeta cells express Cip1 and display substantial hypophosphorylation of Rb but fail to arrest in G(1). Thus, a novel phorbol ester dependent signalling pathway exists in which Cip1 induction is associated with the absence of a G(1) arrest and induction of apoptosis rather than differentiation.

Codeletion of the genes for p16 INK4, methylthioadenosine phosphorylase, interferon-α 1, interferon-β 1, and other 9p21 markers in human malignant cell lines

In this study, 27 malignant cell lines, including leukemias, gliomas, and lung and bladder carcinomas were screened for homozygous deletions of the putative tumor suppressor gene p16 (MTS1/CDK4I/CDKN2) and other markers within the chromosome 9p21 region; these include the genes for interferon-α 1 ( IFNA1), interferon-β 1 ( IFNB1), methylthioadenosine phosphorylase ( MTAP), and two microsatellite markers, D9S171 and D9S169. The purpose of this study was to determine the incidence of codeletion of these markers. Screening for homozygous deletions was carried out using direct polymerase chain reaction of genomic DNA, or, in the case of MTAP, a functional enzyme assay. Of these cell lines, 14 (52%) were found to have homozygous deletions of the p16 gene. Two of the 14 p16-negative cell lines (14%) were found to have homozygous deletions within the p16 domain but no other 9p21 marker. MTAP was codeleted in 12 of the 14 p16-negative cell lines (86%), whereas IFNA1 was codeleted with p16 in eight of these lines (57%); IFNB1 was codeleted in five (36%) of the p16-deleted cell lines. The D9S171 marker, which may lie greater than 3 cM centromeric to p16, is codeleted in three cell lines (21%); the D9S169 marker, which maps even further toward the centromere, was codeleted in only one cell line (7%). Loss of any 9p21 marker, e.g., MTAP or IFNA1, were invariable predictors of the loss of the p16 gene. In addition, loss of IFNA1 always predicted a loss of MTAP (eight of eight cell lines), although loss of MTAP did not always predict a loss of IFNA1 (four of 12 MTAP-deleted cell lines did not have homozygous deletions of IFNA1). Thus loss of nearby genes occurs in a high percentage of cell lines that bear homozygous deletions of the p16 locus. Codeletion of MTAP or IFN in p16-negative malignant cells is of interest, as loss of these genes may influence the biologic behavior of these cells and render them susceptible to certain therapeutic approaches.

Loss of the p16/MTS1 Tumor Suppressor Gene Causes E2F-Mediated Deregulation of Essential Enzymes of the DNA Precursor Metabolism

Homozygous deletions of the tumor suppressor gene p16/MTS1 were reported in a wide variety of tumors and tumor cell lines. Its product inhibits the phosphorylation of the retinoblastoma protein (pRb) by CDK4 and CDK6. Because phosphorylation of pRb is a major regulatory event in the activation of the transcription factor E2F, a role for p16 in the regulation of E2F-dependent transcription was presumed. We investigated the effect of the loss of p16 on E2F-mediated transcription in a tumor progression model consisting of three cell lines originating from a common precursor cell--one p16-positive cell line established from the primary biopsy and two lines derived from more advanced stages of the tumor representing the same cell clone after loss of p16. We observed up- and deregulation of E2F-dependent transcription during the cell cycle of the p16-negative cell clones, which returned to normal after transient expression of p16. This p16-dependent regulation affects a set of enzymes necessary for the activation of all four DNA precursors; it is paralleled by the interconversion of transcriptionally active free E2F and transcriptionally inactive higher molecular complexes of E2F and is dependent on the existence of endogenous pRb. Furthermore, we show that p16-negative cell clones exhibit a growth advantage compared to their p16-positive counterparts. One might speculate that one feature of tumor progression could be deregulation of E2F-dependent transcription caused by loss of p16.

Use of wild-type p53 to achieve complete treatment sensitization of tumor cells expressing endogenous mutant p53.

It is known that transfer of the wild-type p53 gene into p53-negative cells from transgenic mice increases their sensitivity to drug and radiation-induced apoptosis. However, unlike many human tumors, these transgenic cells do not express mutant p53, and it is not known from these earlier studies whether wild-type p53 dominates the effects of mutant p53 with respect to drug and radiation sensitivity. We addressed this question in glioblastoma, a disease characterized by an unusually high level of intrinsic resistance to therapy and poor prognosis: mean survival time from diagnosis is only about 1 yr. We introduced the gene for wild-type p53 into human T98G glioblastoma cells, which express endogenous mutant p53 but not wild-type p53. Stable transfectants that co-expressed mutant and wild-type p53 had enhanced sensitivity to cisplatin and gamma radiation, compared with parental cells, control vector-transduced cells, and transduced cells that had lost expression of wild-type p53. Transient wild-type p53 expression after high-efficiency gene transfer by a p53 adenovirus also sensitized the cells to cisplatin and correlated with the induction of apoptosis. The sensitization effect was also observed in p53 adenovirus-infected H23 small cell lung carcinoma cells, which express endogenous mutant p53. Therefore, wild-type p53 gene transfer has dominant effects over mutant p53 in sensitizing tumor cells to therapy, which supports the potential of p53 gene therapy to enhance the efficacy of traditional therapy.

Cell proliferation patterns and p53 expression in gastric dysplasia.

Gastric dysplasia (high-grade, HGD, and low-grade, LGD) and normal mucosa were tested for anti-p53, anti-Ki-67 and anti-PCNA monoclonal antibodies on paraffin sections, and for relative AgNOR area and number on semithin Epon-Araldite sections. The proliferative compartment in normal mucosa was restricted to the middle layer corresponding to the neck-isthmus region. In LGD and HGD there was an expansion of this compartment to the lower and upper layers of mucosa, and in HGD in particular to the upper layer. p53 was always negative in LGD as well as in normal mucosa, while it was positive in 34 out of 51 cases of HGD. The most discriminant variables between LGD and HGD were relative AgNOR area and the percentages of MIB-1, p53 and PCNA. In p53-positive HGD the highest percentages of PCNA and MIB-1 were in the middle and upper layers (PCNA) or the upper layer (MIB-1), while in p53-negative HGD cases cell proliferation was maximal in the middle layer, although also present in the upper layer. The majority of cases of LGD did not demonstrate cell proliferation in the upper layer, but 5 cases behaved similarly to the p53-negative HGD cases. No significant correlations were found among percentages of MIB-1 and of PCNA and relative AgNOR area and number.

Wild Type p53 Stimulates Expression from the Human Multidrug Resistance Promoter in a p53-negative Cell Line

The effect of human wild type and mutant p53 proteins on the human multidrug resistance (MDR1) promoter was studied in a p53-negative human cell line. Transient expression of MDR1 promoter-chloramphenicol acetyltransferase reporter gene constructs (MDRCAT) cotransfected with p53 expression vectors was analyzed in H358 lung carcinoma cells. Cotransfection with a wild type p53 expression vector stimulated MDRCAT activity, while cotransfection with mutant p53 expression vectors altered at amino acid positions 181, 252, 258, or 273 failed to stimulate expression. Wild type p53 stimulation of MDRCAT activity was time dependent with maximal expression occurring 24-30 h following transfection and correlating with high p53 protein levels. MDR1 promoter deletion analysis suggested that the sequences involved in wild type p53 stimulation of MDRCAT activity were contained within the region from -39 to +53 relative to the start of transcription at +1. This region contains no TATA or p53 consensus binding sequence but does contain an initiator sequence. Wild type p53 stimulation of MDRCAT expression also occurred in parental and doxorubicin-resistant SW620 colon and parental 2780 ovarian cancer cell lines, indicating that wild type p53-mediated simulation of the MDR1 promoter is not restricted to a single cell line.

Apoptosis. Its significance in cancer and cancer therapy.

Apoptosis is a distinct mode of cell death that is responsible for deletion of cells in normal tissues; it also occurs in specific pathologic contexts. Morphologically, it involves rapid condensation and budding of the cell, with the formation of membrane-enclosed apoptotic bodies containing well-preserved organelles, which are phagocytosed and digested by nearby resident cells. There is no associated inflammation. A characteristic biochemical feature of the process is double-strand cleavage of nuclear DNA at the linker regions between nucleosomes leading to the production of oligonucleosomal fragments. In many, although not all of the circumstances in which apoptosis occurs, it is suppressed by inhibitors of messenger RNA and protein synthesis. Apoptosis occurs spontaneously in malignant tumors, often markedly retarding their growth, and it is increased in tumors responding to irradiation, cytotoxic chemotherapy, heating and hormone ablation. However, much of the current interest in the process stems from the discovery that it can be regulated by certain proto-oncogenes and the p53 tumor suppressor gene. Thus, c-myc expression has been shown to be involved in the initiation of apoptosis in some situations, and bcl-2 has emerged as a new type of proto-oncogene that inhibits apoptosis, rather than stimulating mitosis. In p53-negative tumor-derived cell lines transfected with wild-type p53, induction of the gene has, in rare cases, been found to cause extensive apoptosis, instead of growth arrest. Finally, the demonstration that antibodies against a cell-surface protein designated APO-1 or Fas can enhance apoptosis in some human lymphoid cell lines may have therapeutic implications.

P53 protein in low-grade astrocytomas: a study with long-term follow-up.

The immunohistochemical expression of p53 protein (p53) was examined in 52 patients out of a series of 66 patients with low-grade astrocytomas with long-term follow-up. All patients were also evaluated for several clinical and histological features, among which only preoperative Karnofsky score and the extent of surgery were statistically significant parameters to predict outcome on multivariate analysis. p53 accumulation was seen in 46.1% of patients, with a wide range of percentage of positive cells. Median survival for p53-positive and p53-negative patients was 41 and 37 months respectively. The survival curves of p53-positive and -negative patients were not statistically different. However, the curves showed a trend towards a more aggressive course in p53-positive patients beginning 3-4 years after surgery. Five years after diagnosis the survival estimate with the Kaplan-Meier method was 21.2% for patients with p53-positive tumours and 45.9% for patients with p53-negative tumours. This trend is not due to different distribution of major clinical prognostic factors (age, incomplete resection or Karnofsky status). The trend could be related to the time needed by the p53-positive clone to outgrow the rest of the p53-negative neoplastic cell population. This hypothesis is further supported by the fact that the five recurrences which were surgically removed (one anaplastic astrocytoma and four glioblastomas) derived from p53-positive tumours and were themselves intensely p53 positive.

INHIBITION OF CELL-PROLIFERATION BY AN ADENOVIRUS VECTOR EXPRESSING THE HUMAN WILD TYPE-P53 PROTEIN

We have developed human adenovirus 5 (Ad5) vectors expressing the wild type human p53 protein or a mutant p53 form under the control of the human cytomegalovirus immediate early gene promoter. Human cells infected with these vectors expressed high levels of p53, accumulating 20-40 fold more protein than found in normal human fibroblasts. The ability of the vectors to affect proliferation of cells in culture was assessed by measuring cell DNA synthesis and colony forming ability after infection with viruses. When the p53 deficient ovarian carcinoma cell line, SKOV-3, was infected with Adp53wt expressing the wild type (wt) p53 protein, a significant inhibition of cellular DNA synthesis was observed, relative to cells infected with Adp53m expressing mutant p53, or a control virus, AdLacZ, expressing bacterial beta-galactosidase. Inhibition was dependent on multiplicity of infection, with no significant effect below 5 pfu/cell, and maximal effect between 25 and 100 PFU/cell which resulted in approximately 95% inhibition of SKOV-3 cell DNA synthesis relative to mock infected cells. Infection of normal human fibroblasts with Adp53wt also inhibited DNA synthesis but to a significantly lesser degree. SKOV-3 cell survival, assayed by ability to form colonies, was reduced at least 10 fold after infection with Adp53wt compared to colony forming ability of cells after infection with either AdLacZ or Adp53m. The results of these studies indicate that p53 expressed by Ad vectors can inhibit proliferation in culture of p53 negative cells by at least 95%, and suggest that such vectors might similarly inhibit the proliferation of tumor cells in vivo.

Biological activities of p53 mutants in Burkitt's lymphoma cells.

Wild-type human p53 and a series of p53 point mutants isolated from Burkitt's lymphoma (BL) cell lines were tested for their ability to inhibit DNA synthesis in a p53-negative BL cell line and to bind and be degraded by the human papillomavirus type 16 E6 protein. All the mutants lost the wild-type ability to inhibit DNA synthesis, demonstrating that they are all functionally altered. Binding to E6 and consequent degradation of the p53 mutants frequently correlated with changed suppressor properties in BL cells.

Influence of tissue preparation techniques on p53 expression in bronchial and bladder carcinomas, assessed by immunofluorescence staining and flow cytometry

In a series of bronchial and bladder carcinomas, p53 protein expression was examined. Samples from formalin-fixed, paraffin-embedded tissue (routine-treated) were compared with parallel samples of fresh tissue and tissue fixed in paraformaldehyde and ethanol. The expression of p53 was measured by immunofluorescence staining and dual parameter flow cytometry, with simultaneous monitoring of DNA content. For each tumor, p53 fluorescence with different fixatives was expressed relative to fresh tissue. The p53 fluorescence signals were on average brighter from routine-treated tissue than from fresh tissue. The tissue fixed in paraformaldehyde showed no difference from fresh tissue. In the ethanol-fixed tissue, however, fluorescence signals were reduced by nearly 70%, and the fraction of detectable p53 positive cells in tumor tissue was reduced by more than 50%. This loss of fluorescence was probably due to a leakage of the antigen from nucleus to cytoplasm. Pepsin treatment did not influence p53 fluorescence. Within the same tumor, the S-phase fraction in p53 positive cells was significantly higher than in p53 negative cells (13.1 +/- 2.0% vs. 6.5 +/- 0.8%). This pattern was not influenced by formalin fixation or pepsin treatment. Our study demonstrates that in measuring a nuclear antigen, tissue handling may influence the results, and care should be taken to optimize the preparation procedure. Using the antibody PAb 1801, p53 expression measured in archival material is not reduced as compared to fresh tissue.

Expression of Wild-Type p53 Is Not Compatible with - Continued Growth of p53-Negative Tumor Cells

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.

Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.