Search

Infrequent and rare genetic variants in the human population vastly outnumber common ones. Although they may contribute significantly to the genetic basis of a disease, these seldom-encountered variants may also be miss-identified as pathogenic if no correct references are available. Somatic and germline TP53 variants are associated with multiple neoplastic diseases, and thus have come to serve as a paradigm for genetic analyses in this setting. We searched 14 independent, globally distributed datasets and recovered TP53 SNPs from 202,767 cancer-free individuals. In our analyses, 19 new missense TP53 SNPs, including five novel variants specific to the Asian population, were recurrently identified in multiple datasets. Using a combination of in silico, functional, structural, and genetic approaches, we showed that none of these variants displayed loss of function compared to the normal TP53 gene. In addition, classification using ACMG criteria suggested that they are all benign. Considered together, our data reveal that the TP53 coding region shows far more polymorphism than previously thought and present high ethnic diversity. They furthermore underline the importance of correctly assessing novel variants in all variant-calling pipelines associated with genetic diagnoses for cancer.

Mantle cell lymphoma (MCL) is characterized by the hallmark t(11;14)(q13;q32) translocation, leading to cyclin D1 over-expression. Additionally, disrupting the DNA damage response pathway through ATM or TP53 defects plays an important role in MCL pathogenesis. Using deep next-generation sequencing we analyzed the mutual composition of ATM and TP53 mutations in 72 MCL patients, and assessed their impact on progression-free survival (PFS) and overall survival (OS). Mutated ATM and TP53 alleles were found in 51% (37/72) and 22% (16/72) of the cases examined, respectively, with only three patients harboring mutations in both genes. Only a mutated TP53 gene was associated with the significantly reduced PFS and OS and the same output was observed when ATM and TP53 defective groups included also sole deletions 11q and 17p, respectively. Determining the exact ATM/p53 pathway dysfunction may influence the selection of MCL patients for innovative therapies based on the targeted inhibition of selected proteins.

Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).

Mutations and deletions of the tumor suppressor TP53 gene are the most frequent genetic alterations detected in human tumors, though they are rather less frequent in lymphomas. However, acquisition of the TP53 mutation was demonstrated to be one of the characteristic markers in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) and prognostic value of the TP53 status has been recognized for these diseases. We present the complex analysis of the TP53 aberrations in 57 cases of MCL and 131 cases of DLBCL. The TP53 status was determined by functional analyses in yeast (FASAY) followed by cDNA and gDNA sequencing. The level of the p53 protein was assessed by immunoblotting and loss of the TP53-specific locus 17p13.3 was detected by FISH. Altogether, we detected 13 TP53 mutations among MCL cases (22.8%) and 29 TP53 mutations in 26 from 131 DLBCL cases (19.8%). The ratio of missense TP53 mutations was 76.9% in MCL and 82.8% in DLBCL. The frequency of TP53 locus deletion was rather low in both diseases, reaching 9.3% in MCL and 15.3% in DLBCL. The presence of TP53 mutation was associated with shorter overall survival (OS) and progression-free survival (PFS) in MCL. Among DLBCL cases, the TP53 mutations shortened both OS and PFS of patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) and decreased both OS and PFS of patients with secondary DLBCL disease.

D-type cyclins are involved in cell cycle regulation and play an important role in the pathogenesis of lymphomas. Aberrant expression of cyclin D1 is associated with mantle cell lymphoma (MCL) and serves as a diagnostic marker of MCL. Analysis of cyclin D expression in tumor tissues of patients with diffuse large B-cell lymphoma (DLBCL) which comprises a heterogeneous group of tumors may contribute to their stratification. We analyzed expression of cyclin D1, D2, and D3 mRNAs in 30 MCL and 104 DLBCL patients using qRT-PCR and addressed their significance for disease outcome. We confirmed a high level of cyclin D1 mRNA in 29 MCL cases (97%). One case (3%) was identified as positive for cyclin D2. Expression of cyclin D1 was limited to MCL and did not occur in DLBCL. Overexpression of cyclin D2, which is rare in MCL, occurred more frequently in DLBCL (11 cases, 10.6%). We showed that high expression of cyclin D2 in DLBCL cases de novo decreased the overall survival rate (P=0.016) and progression-free survival (P=0.009). The expression pattern of cyclin D3 was similar in both types of studied lymphomas and it did not affect the disease outcome.

Lung cancer is the leading cause of cancer-related deaths worldwide. The p53 tumor suppressor is a transcription factor controlling expression of its target genes in response to various stress stimuli. Mutations of the TP53 gene occur very frequently in lung carcinomas and they play an important role in both oncogenic transformation of lung epithelial cells and lung carcinoma progression. We determined the TP53 status in 42 samples of squamous cell lung carcinoma (SQCC) and 56 samples of lung adenocarcinoma (AC) by the functional analysis FASAY and its variant called split assay. Altogether, we detected 64 TP53 mutations in 63 patients and analyzed them by cDNA and gDNA sequencing. The TP53 mutations were found in 76.2% (32/42) of SQCC cases, and 55.4% (31/56) of ACs. Immunoblotting revealed the p53 protein accumulation in 18 samples (42.9%) among SQCC cases and 19 samples (33.9%) among AC cases. Using fluorescence in situ hybridization we detected loss of the TP53-specific 17p13.3 locus in 23 from 41 analyzed SQCC samples (56.1%) and in 20 from 54 analyzed AC samples (37.0%). We did not find any statistically significant differences in overall and disease-free survival in relation to TP53 status.

The treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) remains a challenging clinical issue. An important treatment option is the use of high-dose corticosteroids. The purpose of this clinical trial was to determine the efficacy and toxicity of an ofatumumab-dexamethasone (O-Dex) combination in relapsed or refractory CLL. The trial was an open-label, multicenter, nonrandomized, Phase II study. The O-Dex regimen consisted of intravenous ofatumumab (Cycle 1: 300 mg on day 1, 2,000 mg on days 8, 15, and 22; Cycles 2-6: 1,000 mg on days 1, 8, 15, and 22) and oral dexamethasone (40 mg on days 1-4 and 15-18; Cycles 1-6). The O-Dex regimen was given until best response, or a maximum of six cycles. Thirty-three patients (pts) were recruited. Twenty-four (73%) pts completed at least three cycles of therapy. The remaining nine pts were prematurely discontinued owing to Grade 3/4 infections (seven pts), disease progression (one pt), or uncontrollable diabetes mellitus (one pt). Overall response rates/complete remissions (ORR/CR) were achieved in 22/5 pts (67/15%). The median progression-free survival (PFS) was 10 months. In pts with p53 defects (n = 8), ORR/CR were achieved in 5/2 pts (63/25%) with a median PFS of 10.5 months. The median overall survival (OS) was 34 months. The Grades 3-5 infectious toxicity in 33% of pts represented the most frequent side effect during the treatment period. In conclusion, the O-Dex regimen shows a relatively high ORR and CR with promising findings for PFS and OS. The study was registered at www.clinicaltrials.gov (NCT01310101)., (© 2015 Wiley Periodicals, Inc.)

TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.

Glioblastoma is the most common and the most aggressive type of brain cancer. Aberrations of the RTK/RAS/PI3K-, p53-, and RB cell signaling pathways were recognized as a core requirement for pathogenesis of glioblastoma. The p53 tumor suppressor functions as a transcription factor transactivating expression of its target genes in response to various stress stimuli. We determined the p53 status in 36 samples of glioblastoma by functional analyses FASAY and split assay. Seventeen p53 mutations were detected and further analyzed by cDNA and gDNA sequencing in 17 patients (47.2 %). Fifteen (88.2 %) of the mutations were missense mutations causing amino acid substitutions, seven of them exhibited temperature-sensitivity. Two mutations were determined as short deletions, one of them causing formation of premature termination codon in position 247. Fluorescent in situ hybridization revealed the loss of the p53-specific 17p13.3 locus in four of 33 analyzed samples (12 %). In 12 out of 30 samples (40 %), the p53 protein accumulation was shown by immunoblotting. There was high (80 %) concordance between the presence of the clonal p53 mutation and the p53 protein accumulation.

The p53 protein is a sequence-specific transcription factor controlling the expression of multiple genes and protecting cells from oncogenic transformation. In many tumors, the p53 protein is completely or partially inactivated by mutations in the p53 gene. We analyzed the transactivating activity of nine human temperature-dependent (td) p53 mutants in yeast cells. Mutations in seven of them were localized in the β-sandwich-coding region of the p53 gene, eight p53 mutants were temperature-sensitive and the R283C mutant was cold-sensitive. Patterns of their transactivation abilities towards three different responsive elements, the extent of their temperature dependency as well as discriminativity, were considerably variable. Similarly, their capacity to become reactivated by amifostine varied from complete resistance to high sensitivity. Transactivation abilities and temperature dependency of six p53 td mutants were determined in transiently-transfected H1299 human cells and revealed substantial concordance between the activity patterns of the p53 mutants in yeast and human cells. We concluded that the td p53 mutants do not comprise a uniform group, therefore, the behavior of each mutant has to be tested individually.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoma in adults. There are specific alterations that appear repeatedly in DLBCL cases and play a role in lymphomagenesis or progression of the disease. Some aberrations were used as prognostic markers in the pre-rituximab era. Addition of rituximab to the classical anthracycline-based chemotherapy significantly increased the survival rate in DLBCL. Only few prognostic factors have been re-evaluated for patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). We performed complex analysis of the p53 tumor suppressor in collection of 75 DLBCL cases. Fifty-four patients were de novo cases, twenty-one cases developed into DLBCL by transformation from less aggressive disease. We determined functional status by analysis of separated alleles in yeast (FASAY) and analyzed the p53 mutations by cDNA sequencing. We assessed the level of the p53 protein by immunoblot analysis. We used FISH to analyze loss of the p53 and ATM (ataxia telangiectasia mutated) gene deletions. We detected 16 p53 mutations (21.3%) including the mutation activating non-sense-mediated RNA decay pathway. Deletion of the p53 allele was more common in cases with p53 mutation. Mutations and/or deletions of p53 had statistically significant negative impact on progression-free survival and tended to decrease also overall survival in 46 de novo DLBCL patients treated with R-CHOP. p53 aberrations are negative predictors for survival of DLBCL patients treated with R-CHOP.

Purpose: There is a distinct connection between TP53 defects and poor prognosis in chronic lymphocytic leukemia (CLL). It remains unclear whether patients harboring TP53 mutations represent a homogenous prognostic group., Patients and Methods: We evaluated the survival of patients with CLL and p53 defects identified at our institution by p53 yeast functional assay and complementary interphase fluorescence in situ hybridization analysis detecting del(17p) from 2003 to 2010., Results: A defect of the TP53 gene was identified in 100 of 550 patients. p53 mutations were strongly associated with the deletion of 17p and the unmutated IgVH locus (both P < .001). Survival assessed from the time of abnormality detection was significantly reduced in patients with both missense (P < .001) and nonmissense p53 mutations (P = .004). In addition, patients harboring missense mutation located in p53 DNA-binding motifs (DBMs), structurally well-defined parts of the DNA-binding domain, manifested a clearly shorter median survival (12 months) compared with patients having missense mutations outside DBMs (41 months; P = .002) or nonmissense alterations (36 months; P = .005). The difference in survival was similar in the analysis limited to patients harboring mutation accompanied by del(17p) and was also confirmed in a subgroup harboring TP53 defect at diagnosis. The patients with p53 DBMs mutation (at diagnosis) also manifested a short median time to first therapy (TTFT; 1 month)., Conclusion: The substantially worse survival and the short TTFT suggest a strong mutated p53 gain-of-function phenotype in patients with CLL with DBMs mutations. The impact of p53 DBMs mutations on prognosis and response to therapy should be analyzed in investigative clinical trials.

Purpose: The aim of this study was to perform a detailed cytogenetic and molecular genetic analysis of a tumor taken from a 14.5-year-old boy with glioblastoma multiforme who showed an atypical clinical course., Methods: Formalin-fixed, paraffin embedded tumor tissue and the corresponding HGG-02 cell line derived from this tumor were analyzed using fluorescence in situ hybridization (FISH), G-banding, multiplex ligation-dependent probe amplification (MLPA), functional analysis of separated alleles in yeast (FASAY), immunohistochemistry (IHC), and immunocytochemistry (ICC)., Results: Mutation of the p53 gene and hypermethylation of the MLH1 gene were detected by FASAY and MLPA, respectively. Cytogenetic analysis showed a polyploid karyotype with extensive heterogeneity in chromosome number. Using FISH, we identified a very unusual genetic change - a loss of EGFR gene copy in both the tumor tissue and the HGG-02 cell line. In accordance with the cytogenetic findings, IHC and ICC did not demonstrate overexpression of EGFR in the tumor tissue or HGG-02 cells., Conclusions: Despite his very poor prognosis, the patient experienced 34 months of event-free survival after surgery and adjuvant radiotherapy and chemotherapy. The detected loss of the EGFR gene copy may contribute to the unusual biological features of this tumor, but the forthcoming detailed expression analysis of cancer regulatory pathways is necessary to better understand this tumor phenotype.

The p53 protein plays an important role in cancer prevention. In response to stress signals, p53 controls essential cell functions by regulating expression of its target genes. Full or partial loss of the p53 function in cancer cells usually results from mutations of the p53 gene. Some of them are temperature-dependent, allowing reactivation of the p53 function in certain temperature. These mutations can alter general transactivation ability of the p53 protein or they modify its transactivation only towards specific genes. We analyzed transactivation of several target genes by 23 temperature-dependent p53 mutants and stratified them into four functional groups. Seventeen p53 mutants exhibited temperature-dependency and discriminative character in human and yeast cells. Despite the differences of yeast and human cells, they allowed similar transactivation rates to the p53 mutants, thus providing evidence that functional analysis of separated alleles in yeast is valuable tool for assessment of the human p53 status.

Mantle cell lymphoma (MCL) is typified by translocation t(11;14)(q13;q32) causing upregulation of cyclin D1 and deregulation of cell cycle. The cyclin D1 activation plays a critical role in MCL pathogenesis but additional oncogenic events, such as aberrations of the ARF/MDM2/p53 pathway are also necessary for progression of the disease. We analyzed the p53 tumor suppressor in tumor tissue of 33 patients with MCL. The p53 status was determined by functional analyses in yeast (FASAY) and by cDNA sequencing. The level of the p53 protein was assessed by immunohistochemistry and immunoblotting. Loss of the p53-specific locus 17p13.3 was detected by FISH. Mutations in the p53 gene were detected in nine samples and they included eight missense mutations and one short deletion causing frame shift and premature stop codon formation in position 169. This mutation was associated with mRNA decay as revealed by sequencing of the p53 gDNA. All eight missense mutations were manifested by accumulation of the p53 protein in nuclei of tumor cells and three of them exhibited loss of the p53-specific locus 17p13.3. The p53 mutations were shown to be a negative prognostic marker in MCL.

Deletion of TP53 gene, under routine assessment by fluorescence in situ hybridization analysis, connects with the worst prognosis in chronic lymphocytic leukemia (CLL). The presence of isolated TP53 mutation (without deletion) is associated with reduced survival in CLL patients. It is unclear how these abnormalities are selected and what their mutual proportion is. We used methodologies with similar sensitivity for the detection of deletions (interphase fluorescence in situ hybridization) and mutations (yeast functional analysis) and analyzed a large consecutive series of 400 CLL patients; a subset of p53-wild-type cases (n = 132) was screened repeatedly during disease course. The most common type of TP53 inactivation, ie, mutation accompanied by deletion of the remaining allele, occurred in 42 patients (10.5%). Among additional defects, the frequency of the isolated TP53 mutation (n = 20; 5%) and the combination of 2 or more mutations on separate alleles (n = 5; 1.3%) greatly exceeded the sole deletion (n = 3; 0.8%). Twelve patients manifested defects during repeated investigation; in all circumstances the defects involved mutation and occurred after therapy. Monoallelic defects had a negative impact on survival and impaired in vitro response to fludarabine. Mutation analysis of the TP53 should be performed before each treatment initiation because novel defects may be selected by previous therapies.

Monitoring of t(14;18) in blood or bone marrow in follicular lymphoma (FL) remains controversial. We attempted to monitor t(14;18) in lymph nodes by ultrasound-guided fine needle aspirations (UG-FNA). First, we confirmed t(14;18) in 27/31 UG-FNAs of lymph nodes with fluorescent in situ hybridisation (FISH) and/or polymerase chain reaction (PCR) in patients with advanced disease. In complete (CR) and molecular remission, there were repeated 18 UG-FNAs in 17 patients. Five of 18 UG-FNA were technically unsuccessful and 6/18 samples contained fibrosis. Despite that, these patients had a better prognosis. In 7/7 aspirations in six patients, t(14;18) was detected. Three patients are still in CR, even one of them remains in long lasting remission despite two consecutive evidences of t(14;18) in UG-FNA. Another three of these patients relapsed a few months after UG-FNA. This study is proof of the principle of the detection of residual t(14;18) bearing cells in previously involved lymph nodes despite patients being in remission.

B-cell chronic lymphocytic leukemia (CLL) is an incurable disease with a highly variable clinical course. A proportion of patients eventually progress to a higher stage of malignancy. A recent association has been observed between the presence of aberrant somatic hypermutations in leukemic cells (hypermutations occurring outside of the immunoglobulin locus) and the transformation to a diffuse large B-cell lymphoma or prolymphocytic leukemia. In this study, we report on the rarely observed blastic transformation in a CLL patient who had previously been shown to harbor aberrant somatic hypermutations in the TP53 tumor-suppressor gene (Mol Immunol 2008;45:1525-29). The enzyme responsible, the activation-induced cytidine deaminase, was still active within the transformation, as evidenced by the ongoing class-switch recombination of cytoplasmic immunoglobulins. The transformation was accompanied by a complete p53 inactivation, as well as complex karyotype changes including prominent amplification of MYCN oncogene. Our case-study supports the view that the aberrant somatic hypermutation is associated with transformation of CLL to a more aggressive malignancy.

Objectives: Abnormalities of the TP53 or ATM, cooperating tumor-suppressor genes, significantly worsen the treatment options for chronic lymphocytic leukemia (CLL) patients. Although the aberrations seem to be mutually exclusive in this leukemia, inactivation of the former gene leads to worse prognosis. We tested the in vitro sensitivity of the CLL samples with heterozygous ATM deletion to fludarabine and combination of fludarabine and rituximab; the responses were compared with the TP53-abnormal and wild-type (wt) cells to delimitate relative significance of ATM deletion., Methods: In vitro analysis was performed on fifty-nine characterized CLL samples using viability assay WST-1. Western blot and real-time RT-PCR were used to monitor the activation of the ATM/p53 pathway., Results and Conclusions: At the clinically relevant concentration of fludarabine, TP53-abnormal samples exhibited markedly higher resistance to fludarabine than the remaining CLL samples (P = 0.012); cohort with ATM deletion was not more resistant than wt cells. A similar induction of the p53 protein and its downstream target genes PUMA and BAX in ATM-deleted and wt cells confirmed that the former subgroup has preserved a critical pro-apoptotic response. Proportions of the samples, which had been sensitized to fludarabine by rituximab pretreatment, were insignificantly lower (P = 0.22) in the TP53-abnormal and ATM-deleted subgroups compared to the wt cases (30%; 29%; 50%, respectively). The presence of ATM (11q22-23) deletion in the CLL cells should not be considered an indication of resistance to fludarabine or its combination with rituximab.

Burkitt's lymphomas (BL) are aggressive rapidly growing tumors typified by a high c-myc expression resulting from t(8;14)(q24;q32), t(2;8)(p12;q24) or t(8;22)(q24;q11) translocations. Alterations of the p53 tumor suppressor are also relatively frequent in BL. Several approaches have been adopted for detection of the p53 aberrations such as immunohistochemical analyses, immunoblotting, DNA sequencing, fluorescence in situ hybridization (FISH), and functional assays. We used these methods to characterize the p53 mutation in tumor cells of a 53-year-old male suffering from Burkitt's lymphoma. By immunohistochemical analyses, we detected high levels of the p53 protein in the tumor tissue. Immunoblotting showed a higher molecular weight of the p53 protein overexpressed in the tumor tissues than that of the standard p53 protein. Similarly, the molecular weight of the PCR product prepared by amplification of the tumor p53 cDNA was higher than that of the standard p53 cDNA. Functional analyses of separated alleles in yeast evidently revealed that the tumor p53 protein was transcriptionally non-functional. The yeast colonies expressing this p53 variant possessed a unique phenotype in that they were red with many white spots on their surface. Sequencing of the tumor cDNA revealed a duplication of the 30 bp region of the p53 gene (g.12155&#95;12184dup30) leading to a repeat of 10 amino acids (Pro-77 to Ala-86) in the p53 protein. Further analyses showed that the mutation was unstable in yeast cells. The FISH analyses did not confer loss of the p53-specific locus 17p13.

Head and neck cancer belongs to the most common types of cancer in both males and females with a mortality rate of approximately 50%. More than 90% of head and neck cancers are squamous cell carcinoma (HNSCC). Carcinogenesis of this disease involves activation of proto-oncogenes and inactivation of tumor suppressor genes. Among them, aberrations of p53 tumor suppressor gene are common events. The aim of this study was to assess the frequency of the tumor suppressor p53 aberrations in Czech population by using a functional test in yeast (FASAY) and by two immunochemical methods. We compared results of the methods and assessed the relationship between the presence of p53 aberration and some clinico-pathological parameters. The following observations were made: i) the accumulated p53 protein was detected in 33 of 50 tested samples (66%) by immunohistochemical analysis and in 27 of 49 tested samples (55.1%) by immunoblotting; ii) the presence of p53 mutation was detected in 36 of 50 tested samples (72%); iii) 6 of 36 p53 mutations detected by FASAY were temperature sensitive (16.7%); iv) 2 independent p53 mutations were found in at least 2 of the 36 positive cases; v) no statistically significant relationship was found between p53 aberration and overall survival.

The tumor suppressor p53 is a transcription factor that participates in control of many cellular functions. All these activities are mediated by direct binding of the p53 tetramer to specific target sequences in promoters of directed genes. Lack of p53 function is often connected with development of cancer, but the frequency of p53 mutations is low in almost all types of leukemia. The aim of this study was to assess the frequency of mutations in the p53 gene in leukocytes of patients with acute myeloid leukemia (AML) using the FASAY functional analysis and to assess the relationship between the presence of p53 mutation and disease outcome. The following observations were made: i) the presence of p53 mutations was detected in 13 of 62 tested AML cases (21%) and in 1 of 4 tested myelodysplastic syndrome (MDS) cases by FASAY; ii) the presence of p53 mutation was shown to be a poor prognostic/predictive factor in AML (p=0.03/0.002); iii) although there is a statistically significant relationship between the presence of p53 mutation and p53 protein accumulation (p=0.05), not all samples having p53 mutation exhibited p53 protein accumulation; iv) five of 13 p53 mutations detected in the leukocytes of AML patients (38.5%) and the mutation detected in the leukocytes of the MDS patient (altogether 6/14-42.9%) were partially inactivating ts mutations. The high frequency of the ts p53 mutations in our study; and a novel modification in performing FASAY, are discussed; v) different ts mutations differ in the level of their temperature sensitivity and in their responsiveness to the cytoprotective drug amifostine.

Proteins within the Rel/NF-kappa B transcription factor family can be divided into two functional domains, a homologous amino terminal region, the Rel Homology Domain, and a divergent carboxy terminal domain. The amino terminal sequences specify DNA binding, nuclear localization, and interaction with the I kappa B family of inhibitory proteins. The carboxy terminus of each protein functions as a transcriptional activation domain, however, precise definition of sequence requirements has been difficult. To further define these sequences, small 100 bp deletions were constructed throughout the carboxy terminus of v-Rel. Each resulting mutant was assayed for DNA binding, localization, protein complex formation, activation of endogenous gene expression and ability to transform bone marrow cells and fibroblasts. Surprisingly, deletion within the carboxy terminus had marginal effects on transforming potential. However, three separate regions were required for full activation of gene expression. Taken together, these results suggest that the carboxy terminus of v-Rel contains multiple sequences that participate in the activation of gene expression.

To determine the physiological processes in which the transcription factor c-Rel may act, we have examined its pattern of expression in the avian embryo by in situ hybridization. These studies showed that c-rel is expressed ubiquitously at low levels and at high levels in isolated cells undergoing programmed cell death by apoptosis or autophagocytosis. To further establish a functional link between expression of c-rel and cell death, we examined the biological consequences of c-rel overexpression in vitro. In primary avian fibroblasts, overexpression of c-rel leads to transformation and dramatic life span extension. In contrast, bone marrow cells expressing high levels of c-rel undergo a process of programmed cell death displaying features of both apoptosis and autophagocytic cell death. Thus, these experiments suggest a critical role for c-rel not only in the control of cell proliferation, but also in the induction of cell death.