Loss Of Wild-type Allele Research Articles

Gain of function of mutant p53 by coaggregation with multiple tumor suppressors

Many p53 missense mutations possess dominant-negative activity and oncogenic gain of function. We report that for structurally destabilized p53 mutants, these effects result from mutant-induced coaggregation of wild-type p53 and its paralogs p63 and p73, thereby also inducing a heat-shock response. Aggregation of mutant p53 resulted from self-assembly of a conserved aggregation-nucleating sequence within the hydrophobic core of the DNA-binding domain, which becomes exposed after mutation. Suppressing the aggregation propensity of this sequence by mutagenesis abrogated gain of function and restored activity of wild-type p53 and its paralogs. In the p53 germline mutation database, tumors carrying aggregation-prone p53 mutations have a significantly lower frequency of wild-type allele loss as compared to tumors harboring nonaggregating mutations, suggesting a difference in clonal selection of aggregating mutants. Overall, our study reveals a novel disease mechanism for mutant p53 gain of function and suggests that, at least in some respects, cancer could be considered an aggregation-associated disease.

Mutation Analysis of the APC Gene in a Chinese FAP Pedigree with Unusual Phenotype

Background and Aim. Germline mutations of the adenomatous polyposis coli (APC) gene cause familial adenomatous polyposis (FAP), an autosomal dominant inherited disease mainly characterized by colorectal adenomatous polyposis. Genetic studies of FAP have shown that somatic APC mutations are dependent on the position of the germline APC mutation. However, the molecular mechanism underlying these genotype-phenotype associations for APC in Chinese remain largely unknown. Patients and Methods. In this study, we investigated the APC gene mutation in a Chinese FAP family by systematic screening with multiplex ligation-dependent probe amplification (MLPA), denaturing high-performance liquid chromatography (dHPLC), and DNA sequencing. Promoter methylation was detected by methylation-specific PCR. Results. The identical germline mutation c.1999 C>T (Q667X) of APC was identified in 5 affected members, among which 2 members carried somatic mutations of APC, one with promoter hypermethylation and the other with loss of wild-type allele in their adenomas. The somatic mutations were shown connected with the disease severity, demonstrating a unique genotype-phenotype association in this FAP pedigree. Conclusion. The study revealed the existence of novel pathogenic mutations in Chinese patients with FAP. Somatic mutations are of particular interest because of the unusual phenotypic features shown by patients.

Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells

BackgroundActivating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.Methodology/Principal FindingsWe determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival.ConclusionsMASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

Dextran sodium sulfate strongly promotes colorectal carcinogenesis in ApcMin/+ mice: Inflammatory stimuli by dextran sodium sulfate results in development of multiple colonic neoplasms

The mouse model for familial adenomatous polyposis, Apc(Min/+) mouse, contains a truncating mutation in the Apc gene and spontaneously develops numerous adenomas in the small intestine but few in the large bowel. Our study investigated whether dextran sodium sulfate (DSS) treatment promotes the development of colonic neoplasms in Apc(Min/+) mice. Apc(Min/+) and Apc+/+ mice of both sexes were exposed to 2% dextran sodium sulfate in drinking water for 7 days, followed by no further treatment for 4 weeks. Immunohistochemistry for cyclooxygenase-2, inducible nitric oxide synthase, beta-catenin, p53, and nitrotyrosine, and mutations of beta-catenin and K-ras and loss of wild-type allele of the Apc gene in the colonic lesions were examined. Sequential observation of female Apc(Min/+) mice that received DSS was also performed up to week 5. At week 5, numerous colonic neoplasms developed in male and female Apc(Min/+) mice but did not develop in Apc+/+ mice. Adenocarcinomas developed in Apc(Min/+) mice that received DSS showed loss of heterozygosity of Apc and no mutations in the beta-catenin and K-ras genes. The treatment also significantly increased the number of small intestinal polyps. Sequential observation revealed increase in the incidences of colonic neoplasms and dysplastic crypts in female Apc(Min/+) mice given DSS. DSS treatment increased inflammation scores, associated with high intensity staining of beta-catenin, cyclooxygenase-2, inducible nitric oxide synthase and nitrotyrosine. Interestingly, strong nuclear staining of p53 was specifically observed in colonic lesions of Apc(Min/+) mice treated with DSS. Our results suggest a strong promotion effect of DSS in the intestinal carcinogenesis of Apc(Min/+) mice. The findings also suggest that strong oxidative/nitrosative stress caused by DSS-induced inflammation may contribute to the colonic neoplasms development.

Loss of heterozygosity of chromosome 12p does not correlate with KRAS mutation in non-small cell lung cancer.

Activating mutations of RAS gene families have been found in a variety of human malignancies, including lung cancer, suggesting their dominant role in tumorigenesis. However, several studies have shown a frequent loss of the wild-type KRAS allele in the tumors of murine models and an inhibition of oncogenic phenotype in tumor cell lines by transfection of wild-type RAS, indicating that wild-type RAS may have oncosuppressive properties. To determine whether loss of wild-type KRAS is involved in the development of human lung cancer, we investigated the mutations of KRAS, NRAS and BRAF in 154 primary non-small cell lung cancers (NSCLCs) as well as 10 NSCLC cell lines that have been shown to have KRAS mutations. We also determined the loss of heterozygosity status of KRAS alleles in these tumors. We detected point mutations of KRAS in 11 (7%) of 154 NSCLCs, with 10 cases at codon 12 and 1 at codon 61, but no mutations of NRAS or BRAF were found. Using the laser capture microdissection technique, we confirmed that 9 of the 11 tumors and 7 of the 10 NSCLC cell lines retained the wild-type KRAS allele. Among the cell lines with heterozygosity of mutant and wild-type KRAS, all of the cell lines tested for expression were shown to express more mutated KRAS than wild-type mRNA, with higher amounts of KRAS protein also being expressed compared to the cell lines with a loss of wild-type KRAS allele. In addition, among 148 specimens available for immunohistochemical analysis, 113 (76%) showed positive staining of KRAS, indicating that the vast majority of NSCLCs continue to express wild-type KRAS. Our findings indicate that the wild-type KRAS allele is occasionally lost in human lung cancer, and that the oncogenic activation of mutant KRAS is more frequently associated with an overexpression of the mutant allele than with a loss of the wild-type allele in human NSCLC development.

Aspects of tuberous sclerosis complex (TSC) protein function in the brain.

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumour-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at both TSC1 and TSC2 loci. LOH has been documented in renal angiomyolipomas, but loss of the wild-type allele in cortical tubers appears very uncommon. We analysed 24 hamartomas from 10 patients for second-hit mutations by several methods, and found no evidence for the inactivation of the second allele in many of the central nervous system (CNS) lesions, including tumours that appear to be clonally derived. We believe that somatic mutations in TSC1 and TSC2 resulting in the loss of wild-type alleles may not be necessary in some tumour types, and other mechanisms may contribute to tumorigenesis in this setting. We have shown that hamartin interacts with neurofilament light chain (NF-L) and could integrate the neuronal cytoskeleton through its direct interaction with NF-L and ERM (ezrin/radixin/moeisin) proteins. Our unpublished work further documents the binding of tuberin with Pam, a protein associated with c-Myc, which is enriched in brain. All these observations suggest that the tuberin-hamartin complex is likely to have distinct functions in the CNS.

Genetic and clinical characterization of sporadic cystic parathyroid tumours.

The hyperparathyroidism--jaw tumour (HPT--JT) syndrome is one of the familial disorders characterized by primary hyperparathyroidism and has been linked to the chromosomal region of 1q32--q21. The parathyroid tumours related to this syndrome have shown loss of wild-type alleles at this locus suggesting that inactivation of a tumour suppressor gene might be responsible for the disease. In the majority of these tumours cysts are a prominent feature. By loss of heterozygosity (LOH) studies, we investigated the region of interest in an attempt to clarify its possible role in a series of cystic sporadic parathyroid adenomas. A total of 30 patients diagnosed with sporadic hyperparathyroidism were included in the study, genotyped with 17 polymorphic microsatellite markers at chromosome 1q, and additional markers from 1p and 11q13 which are commonly involved in sporadic parathyroid tumours. The cystic parathyroid tumours were characterized clinically, and immunohistochemistry against PTH was carried out to confirm the parathyroid origin of the cysts. LOH was found in six of 30 tumours (20%) on 1q, six of 30 tumours (20%) on 1p and five of 30 tumours (17%) on 11q13. We found a significant correlation between allelic alterations and the clinical parameters, tumour weight and PTH. Furthermore, we found a significant difference between tumour weight and PTH in cases of cystic parathyroid tumours compared with unselected sporadic cases. These results suggest that cystic parathyroid tumours might represent a new subgroup among parathyroid tumours based on the genetic and clinical findings. Loss of heterozygosity at 1q further supports the presence of a tumour suppressor gene at this locus.

The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials

AbstractIn acute myeloid leukemia (AML), further prognostic determinants are required in addition to cytogenetics to predict patients at increased risk of relapse. Recent studies have indicated that an internal tandem duplication (ITD) in the FLT3 gene may adversely affect clinical outcome. This study evaluated the impact of a FLT3/ITD mutation on outcome in 854 patients, mostly 60 years of age or younger, treated in the United Kingdom Medical Research Council (MRC) AML trials. An FLT3/ITD mutation was present in 27% of the patients and was associated with leukocytosis and a high percentage of bone marrow blast cells (P < .001 for both). It had a borderline association with a lower complete remission rate (P = .05) and a higher induction death rate (P = .04), and was associated with increased relapse risk (RR), adverse disease-free survival (DFS), event-free survival (EFS), and overall survival (OS) (P < .001 for all). In multivariate analysis, presence of a mutation was the most significant prognostic factor predicting RR and DFS (P < .0001) and was still significant for OS (P = .009) and EFS (P = .002). There was no evidence that the relative effect of a FLT3/ITD differed between the cytogenetic risk groups. More than one mutation was detected in 23% of FLT3/ITD+ patients and was associated with worse OS (P = .04) and EFS (P = .07). Biallelic disease or partial/complete loss of wild-type alleles was present in 10% of FLT3/ITD+ patients. The suggestion is made that detection of a FLT3/ITD should be included as a routine test at diagnosis and evaluated for therapeutic management.

Survey of Somatic Mutations in Tuberous Sclerosis Complex (TSC) Hamartomas Suggests Different Genetic Mechanisms for Pathogenesis of TSC Lesions

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumor-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at loci of both TSC1 and TSC2. LOH has been documented in renal angiomyolipomas (AMLs), but loss of the wild-type allele in cortical tubers appears to be very uncommon. Analysis of second, somatic events in tumors for which the status of both TSC1 and TSC2 is known is essential for exploration of the pathogenesis of TSC-lesion development. We analyzed 24 hamartomas from 10 patients for second-hit mutations, by several methods, including LOH, scanning of all exons of both TSC1 and TSC2, promoter methylation of TSC2, and clonality analysis. Our results document loss of the wild-type allele in six of seven AMLs, without evidence of the inactivation of the second allele in many of the other lesions, including tumors that appear to be clonally derived. Laser-capture microdissection further demonstrated loss of the second allele in all three cellular components of an AML. This study thus provides evidence that, in both TSC1 and TSC2, somatic mutations resulting in the loss of wild-type alleles may not be necessary in some tumor types-and that other mechanisms may contribute to tumorigenesis in this setting.

A new functional classification of tumor-suppressing genes and its therapeutic implications.

Cell fusion studies have demonstrated that malignancy can be suppressed by a single dose of malignancy suppressor genes (MSGs), indicating that malignancy is a recessive phenotype. Correspondingly, it is widely believed that mutational inactivation of both alleles of tumor suppressor genes (TSGs), in familial and sporadic tumors, is the formal proof of the recessive nature of malignancy. Evidence presented here, however, shows that unlike MSGs, identified solely through cell fusion studies with no gene of this class yet cloned, many well-known TSGs have gene dosage effects and inhibit cellular growth in vitro. Moreover, homozygous inactivation of a growth-inhibitory TSG (GITSG) is not directly correlated with malignancy. An alternative interpretation is provided for the loss of wild-type alleles of these genes in the tumors. It is concluded that the MSGs and the GITSGs do not belong to the same class of genes. The functional classification of tumor-suppressing genes has important implications for developing effective cancer therapies.

Genetic alterations in 'normal' luminal and myoepithelial cells of the breast.

Chromosomal loci exhibiting loss of heterozygosity (LOH) at high frequency in invasive breast cancer have been investigated in 'normal' breast tissue from patients with carcinoma and from reduction mammoplasty specimens. Duct-lobular units dissected from paraffin-embedded tissues and 485 'normal' luminal and myoepithelial cell clones were studied. Overall, LOH was found in normal cells in 5/10 breast cancer cases and 1/3 reduction mammoplasty specimens. LOH was identified in normal cells adjacent to and distant from the tumour. In one case, all luminal and myoepithelial samples exhibited loss of the same allele on chromosome 13q. One case in which the patient had a germline truncating mutation in the BRCA1 gene exhibited LOH on 17q in 3/33 normal clones. One of these clones showed loss of wild-type allele indicating gene inactivation. This sample also had LOH at markers on chromosomes 11p and 13q. One of 93 clones from three reduction mammoplasties showed allele loss at a locus on chromosome 13q. The identification of LOH in breast lobules suggests that they may be clonal. The demonstration of genetic alteration in luminal and myoepithelial cells provides evidence for the presence of a common stem cell for the two epithelial cell types. LOH has been demonstrated in normal tissues near and away from the carcinoma, suggesting that genetic alterations are likely to be more heterogeneous and widespread than is currently envisaged, and probably occur very early in breast development. Homozygous deletion of BRCA1 per se does not appear to provide clonal advantage.

TP53 DNA contact mutations are selectively associated with allelic loss and have a strong clinical impact in head and neck cancer.

Recent studies have suggested that different mutation types within the core domain of the tumour suppressor protein p53, i.e. DNA contact mutations and structural mutations, confer different biological properties. We have analysed in 86 head and neck squamous cell carcinomas (HNSCC), whether these p53 mutation types have a differential clinical impact. Thirty-seven missense mutations were identified. Thirteen of these (36%) were DNA contact mutations, occurring in the L3 loop, in the H2 loop sheet helix motif, in the S10 beta strand and in Zinc binding residues. Microsatellite marker analysis revealed a selective association between these mutations and the loss of wild-type alleles (100% LOH vs 50% LOH in tumours with structural mutations; P=0.0034, Fisher's exact, 2-tailed). In comparison to structural mutations or to the absence of mutations in the core domain, DNA contact mutations were associated with higher tumour stages (84.6% vs 62%), a higher incidence of lymph node metastasis (91.7% vs 56%; P=0.014, Fisher's exact, 2-tailed), a shortened recurrence-free survival (8.1 months vs 23.7 months, P=0.047, log rank test) and overall survival (11 months vs 29.2 months; P=0.003, log rank test). The latter was also the case when only stage IV tumours were analysed (P=0.0055, log rank test). These data indicate that in HNSCC, TP53 DNA contact mutations confer a strong selection pressure to eliminate wild-type alleles, and that they result in an accelerated tumour progression and reduced therapeutic responsiveness.

K-ras mutations and allelic loss at 5q and 18q in the development of human pancreatic cancers.

Our findings have implications for early diagnosis and for the identification of patients at increased risk. Invasive cancers of the pancreas frequently are preceded by and associated with a spectrum of preneoplastic changes. We investigate the presence of K-ras mutations and allelic loss at 5q and 18q loci in preneoplastic lesions associated with nine cases of invasive pancreatic ductal carcinomas. We precisely microdissected 115 foci of normal, preinvasive, and invasive foci from paraffin-embedded sections. 1. K-ras mutations occur early in the pathogenesis of pancreatic adenocarcinoma. Mutations were identified in multiple preneoplastic foci associated with al six cases in which ras mutations were present in the corresponding invasive cancers, including nearly all foci of mucous cell and atypical hyperplasia, in some cases of papillary hyperplasia (40%), and in one example of morphologically normal epithelium. 2. Ras mutations in preneoplastic foci are widespread, occur distant from the invasive tumor, and may present multiple mutations. Two, and in one case three, different types of K-ras mutations were found in separate preneoplastic foci from three individual cases. 3. Evidence for a "second hit" in the ras gene (i.e., loss of wild-type allele or amplification of the mutant allele) was present in some tumors and may be associated with the invasive process. 4. In contrast to ras mutations, limited data suggest that loss of heterozygosity (LOH) at 5q and 18q are relatively late events.

R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata

Site-directed mutagenesis of the photosynthetic apparatus (PSA) genes in Rhodopseudomonas capsulata is presented utilizing a transposon Tn7 mutagenized R-prime. The R-prime, pRPS404, bears most of the genes necessary for the differentiation of the photosynthetic apparatus. Mutagenesis of the R-prime with Tn7 in Escherichia coli, conjugation into R. capsulata, and homologous recombination with the wild-type alleles efficiently generates photosynthetic apparatus lesions. Wild-type alleles are lost spontaneously and the Tn7-induced lesions are revealed by subsequent intramolecular recombination between IS21 insertion elements that bracket the prime sequences in direct repeat. The molecular nature of the intermediates involved in the transposition, recombination and deletion have been investigated by Southern hybridization analysis. The spontaneous loss of wild-type alleles after homologous recombination with the chromosome may be of general use to other prokaryotic site-directed transposon mutagenesis schemes. The IS21-mediated deletion of the prime DNA is dependent on the RecA protein in E. coli, generating the parental R-factor bearing one IS21 element. A genetic-physical map exists for a portion of the prime photosynthetic apparatus DNA. When Tn7 is inserted into a bacteriochlorophyll gene in the Rprime and then crossed into R. capsulata, mutants are produced that accumulate a bacteriochlorophyll precursor, which is in excellent agreement with the existing genetic-physical map. This corroborates our mutagenesis scheme. Mutants arising from Tn7 insertions outside of the genetic map have been isolated. Light harvesting II mutations have been isolated; one mutant lacks only the 14,000 M r, polypeptide.