PTEN (MMAC1) mutations are frequent in primary glioblastomas (de novo) but not in secondary glioblastomas

Loss of heterozygosity (LOH) on chromosome 10 is the most frequent genetic alteration associated with the evolution of malignant astrocytic tumors and it may involve several loci. The tumor suppressor gene PTEN (MMAC1) on chromosome 10q23 is mutated in approximately 30% of glioblastomas (WHO Grade IV). In this study, we assessed the frequency of PTEN mutations in primary glioblastomas, which developed clinically de novo, and in secondary glioblastomas, which evolved from low-grade (WHO Grade II) or anaplastic astrocytomas (WHO Grade III). Nine of 28 (32%) primary glioblastomas contained a PTEN mutation and an additional case showed a homozygous PTEN deletion. This indicates that after overexpression/amplification of the EGF receptor, loss of PTEN function is the most common alteration in primary glioblastomas. In this series, 5 of 28 (18%) primary glioblastomas showed both a PTEN mutation and EGFR amplification. In contrast, only 1 of 25 (4%) secondary glioblastomas contained a PTEN mutation, and none of them showed a homozygous PTEN deletion. The secondary glioblastoma with a PTEN mutation developed from an anaplastic astrocytoma that already carried the mutation. The observation that secondary glioblastomas have a p53 mutation as a genetic hallmark but rarely contain a PTEN mutation supports the concept that primary and secondary glioblastomas develop differently on a genetic level.     

Overexpression and mutations of p53 in metastatic malignant melanomas

Heterocypris incongruens (RAMD.) was selected as an intermediate host, having emerged as the main host for Diorchis stefanskii Czapl. in experimental conditions. It was found that the index of infective activity for larvae of D. stefanskii was high on the first day, amounting to 50-54%, with an extensity of infection of 100%. The infective activity of larvae declined slowly but steadily at the low temperature (5 degrees C). Oncospheres retained their infective activity for more than 2.5 months. In contrast, in culture at 18-20 degrees C and 25 degrees C, the infective activity of larvae had declined rapidly only a few days into the experiment. The irrevocable loss of tapeworm infectivity occurred at temperatures of -5 degrees C and +38 degrees C, at which 100% of oncospheres died.     

Overexpression of the p53 gene in primary and metastatic head and neck carcinomas

The p53 gene has been correlated with disease progression in a number of human malignancies, and p53 abnormalities are found in a high percentage of head and neck squamous cell carcinomas. The objectives of this study were 1. to correlate p53 expression with disease progression in squamous cell carcinoma of the head and neck (SCCHN), and 2. to determine whether there are site-specific differences in p53 expression. Primary lesions and/or lymph node metastases from 147 patients with invasive SCCHN were immunostained for p53 overexpression. Expression of p53 was similar (42% versus 43%) in primary lesions and lymph node metastases. Expression also did not vary significantly by site in the head and neck. In conclusion, increased p53 expression did not correlate with disease progression in our series of patients with invasive SCCHN. The finding of a lack of increased expression with disease spread to lymph nodes supports the belief that p53 alterations occur early in head and neck carcinogenesis.     

Cell elongation and septation are two mutually exclusive processes in Escherichia coli

Bacterial rod morphogenesis was studied in synchronously growing cells of Escherichia coli C600 during the reshaping process that follows the removal of mecillinam, a beta-lactam antibiotic that specifically inhibits lateral wall formation of gram-negative rods and causes transition to coccal shape. Removal of mecillinam after 30 min of action did not affect the timing of subsequent cell division, but removal after 50 min delayed resumption of cell division for approximately one generation time. In order to study the interplay between lateral wall elongation and septum formation in determining and maintaining the bacterial rod shape, we evaluated the effect of re-adding mecillinam or of adding aztreonam (a specific inhibitor of septum formation) at various stages of the reshaping process. We conclude that mecillinam was active only during the reshaping process, while aztreonam was active only later when the cells were close to dividing again. These results provide further evidence for our previous proposal according to which elongation and septation are two alternating and competing events of the cell cycle and are linked to each other to force bacterial rods to grow to a given length.     

WIN 35,428 and mazindol are mutually exclusive in binding to the cloned human dopamine transporter

It has been suggested that cocaine and mazindol bind to separate sites on the dopamine transporter. In the present study, we address this issue by examining the inhibition by mazindol of the binding of [3H]WIN 35,428 ([3H]2beta-carbomethyoxy-3beta-(4-fluorophenyl)-tropane), a phenyltropane analog of cocaine, and the inhibition by WIN 35,428 of [3H]mazindol binding to the cloned human dopamine transporter expressed in C6 glioma cells. The design involved the construction of inhibition curves at six widely different radioligand levels, enabling the distinction between the nonlinear hyperbolic competition (i.e., negative allosteric) model and the competitive (i.e., mutually exclusive binding) model. Nonlinear computer curve-fitting analysis indicated no difference in the goodness of fit between the two models; the negative allosteric model indicated an extremely high allosteric constant of approximately > or = 100, which practically equates to the competitive model. The present results suggest that complex interactions reported between cocaine and mazindol in inhibiting dopamine transport are beyond the level of ligand recognition.     

Apoptosis and expression of inducible nitric oxide synthase are mutually exclusive in renal mesangial cells

Nitric oxide (NO) is a multipurpose messenger molecule, important for blood vessel relaxation, neuronal communication, and antimicrobial activities. The generation of NO from L-arginine is catalyzed by NO synthase (NOS). An inducible form of NOS, iNOS, was first characterized in macrophages and then in many other tissues and cells, including renal mesangial cells. Mesangial cells play a crucial role in the regulation of the glomerular filtration rate as well as in the pathophysiology of certain forms of glomerulonephritis in which mesangial cells and macrophages produce NO in high amounts. Because reports have associated NO production with apoptotic cell death in macrophages and we recently demonstrated NO-mediated apoptosis in mesangial cells, we searched for the relationship between in situ iNOS induction and apoptosis by iNOS immunocytochemistry and terminal desoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. RAW 264.7 macrophages exhibited homogeneous iNOS expression and apoptotic nuclei in the iNOS-containing cells upon stimulation with interferon-gamma and lipopolysaccharide. In contrast, stimulated rat mesangial cells stained heterogeneously for iNOS, depending on cell passage and iNOS-stimulating pathway. Mesangial cells expressing iNOS did not display signs of apoptosis and, vice versa, cells showing characteristic features of apoptosis did not stain for iNOS. Thus, our study suggests that mesangial cells react to stimulation by interleukin-1 and/or cAMP-elevating compounds with mutually exclusive responses, either by expression of iNOS or by undergoing programmed cell death.     

Domains of retinoid signalling and neurectodermal expression of zebrafish otx1 and goosecoid are mutually exclusive

Retinoid signalling plays an important role in embryonic pattern formation. Excess of retinoic acid during gastrulation results in axial defects in vertebrate embryos, suggesting that retinoids are involved in early anteroposterior patterning. To study retinoid signalling in zebrafish embryos, we developed a novel method to detect endogenous retinoids in situ in embryos, using a fusion protein of the ligand inducible transactivation domain of a retinoic acid receptor and a heterologous DNA binding domain. Using this method, we show that retinoid signalling is localized in zebrafish embryos in the region of the embryonic shield, and towards the end of gastrulation in a posterior dorsal domain. To investigate the relationships between the spatial distribution of retinoid signalling and the regulation of retinoid target genes, we studied the downregulation by retinoic acid of two genes expressed in anterior regions of the embryo, goosecoid and otx1. These experiments show that expression of both genes is strongly downregulated in the anterior neurectoderm of zebrafish embryos treated with retinoic acid, whereas mesendodermal expression is only mildly affected. Interestingly, a significant downregulation of goosecoid expression by retinoic acid was observed only during midgastrulation but not in earlier stages. In agreement with these results, spatial expression of goosecoid and otx1 does not overlap with the region of retinoid signalling in the late gastrula. Our data support the hypothesis that a localized retinoid signal is involved in axial patterning during early development, at least in part through the repression of anterior genes in posterior regions of the embryo. Furthermore, our data suggest that the action of retinoids is spatially as well as temporally regulated in the developing embryo.     

p53 mutations in breast cancer: incidence and relations to tumor aggressiveness and evolution of the disease

Breast cancer is a polymorphic disease and, until now, nodal invasion and steroid receptor levels remain the most powerful and widely used prognostic indicators. Molecular oncology has proven the importance of somatic genetic events in cancer genesis and evolution. In breast cancer a number of genetic aberrations have been proposed to bear impact on disease outcome. Greatest significance has been associated to ERBB2 amplification and overexpression. More recently p53 mutations have been suggested to bear meaning in terms of cancer evolution. We discuss here the molecular epidemiology of p53 mutations in human breast tumors and the clinico-pathological significance that can be associated to them.     

Primary human prostate cancer cells harboring p53 mutations are clonally expanded in metastases

Recent studies suggest a role for p53 in prostate cancer progression. Although p53 mutations in primary prostate cancer tissues are relatively infrequent, they occur at significant levels in metastatic disease. Here we describe a novel approach to the molecular analysis of p53 in paired specimens of primary and metastatic prostate cancer that results in quantitative estimates of the extent of clonal expansion. In 20 pairs with 1 or both specimens p53 immunopositive and in 6 pairs with both specimens immunonegative, the frequency of mutations was estimated by microdissection of the cancer from fixed and sectioned tissues, isolation of the DNA followed by PCR amplification of p53 genomic fragments, and cloning of the PCR products into plasmid vectors. At least 90 clones/tissue specimen were screened for mutations by single-strand conformational polymorphism analysis. DNA from abnormally migrating single-strand conformational polymorphism samples was sequenced to confirm mutations. Missense mutations in exon 5, 7, or 8 were detected in 9 of 20 immunopositive pairs and in 1 of 6 immunonegative pairs. A marked heterogeneity of mutations in primary prostate cancer was apparent. The frequency of p53 mutations was greater in the metastases than in the primary tumors. In three immunopositive pairs, the same p53 mutation was demonstrated at a low frequency in the primary tumor but was demonstrated at a greater frequency in the metastasis, indicating relatively limited clonal expansion of cells harboring specific p53 mutations in the primary tumor, yet significant clonal growth at metastatic sites as determined by this novel method.     

p53 mutations and overexpressions in Japanese breast cancer

In this paper, we explore the use of a variety of familial transmission tests (and case-control analyses) to screen for allelic associations in simulated marker data of a quality (2 cM map) that will feasibly arise from genomic scans within the next 5-10 years. We demonstrate a form of the transmission-disequilibrium test extended to multiallele systems. The methods used were log-linear and related models implemented largely using standard statistical packages.     

Low frequency of the p53 gene mutations in neuroblastoma

BACKGROUND: The p53 gene frequently is affected by point mutations, rearrangements, or deletions that contribute to the genesis or progression of a wide variety of human adult solid tumors; however, to the authors' knowledge, this gene alteration has not been analyzed in neuroblastoma. METHODS: Genomic DNA samples from 20 children with neuroblastoma, including 16 patients with advanced disease, were screened for the presence of mutations in exons 5-9 of the p53 gene, where over 90% of mutations have been reported to be located in human cancer. The screening technique employed polymerase chain reaction/single-strand conformation polymorphism analysis followed by direct DNA sequencing. RESULTS: Heterozygous mutations were detected in 2 of the 20 cases. A silent mutation (T to G transversion) at codon 172 and a missense mutation (G to T transversion) at codon 259 were found in patients with Stage II and Stage IV disease, respectively. Thus, p53 mutations were found to occur in neuroblastoma, but at a low frequency (2 of 20). CONCLUSIONS: Our data suggest that in a minority of neuroblastomas, p53 gene mutations may play a contributing role in tumorigenesis, but other genes presumably play a major role in this tumor.     

K-ras and p53 mutations are an independent unfavourable prognostic indicator in patients with non-small-cell lung cancer

We examined 159 consecutive cases of non-small-cell lung cancer (NSCLC) for a mutation at codon 12 of the K-ras gene and for a mutation of the p53 gene occurring in exons 5-8. Eleven (6.9%) had mutations of the K-ras (ras+) and 57 (35.8%) had mutations of the p53 (p53+). There were 95 cases (59.7%) with ras- p53-, seven cases (4.4%) with ras+/p53-, 53 cases (33.3%) with ras-/p53+ and four cases (2.5%) with ras+/p53+. The ras+ group had a worse prognosis than the ras group in all cases and in 107 early-stage cases (stage I-II, P<0.05). The p53+ group had a worse prognosis in 107 early-stage cases (P<0.01), but there was no statistically significant difference when 52 advanced-stage cases (stage III-IV) or all patients were considered. Both ras and p53 mutations were unfavourable prognostic factors in 94 cases with adenocarcinoma, but there was no statistical significance in 57 cases with squamous cell carcinoma. According to Cox's model, the pathological stage, ras mutation and p53 mutation were found to be independent prognostic factors. Our results suggest that ras and p53 mutations were independent unfavourable prognostic markers especially in the early stage of NSCLC or in adenocarcinoma.     

p53 gene mutations in osteosarcomas of low-grade malignancy

Alterations in tumor suppressor gene p53, localized on chromosome 17p13, are considered to play a significant role in the initiation and, to some extent, even in the progression of various malignant tumors. In this respect, investigations on conventional highly malignant osteosarcomas have shown a mutation rate of approximately 20%. However, currently, data on the mutation rate in the group of variant histology osteosarcomas of low-grade malignancy do not exist. Therefore, we investigated a panel of low malignant entities (five low malignant intramedullary osteosarcomas grade 1; one intramedullary osteosarcoma grade 2; eight parosteal osteosarcomas, including one local recurrence grades 1 and 2, and five periosteal osteosarcomas grade 2) with polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis focusing on exons 4 to 8 of the p53 gene followed by direct sequencing. Point mutations were found in one low-grade osteoblastoma-like osteosarcoma and in two periosteal osteosarcomas grade 2 (one missense, one silent, and one nonsense mutation). This mutation rate of 15.7% (3 of 19) is comparable to that determined in highly malignant osteosarcomas. Moreover, the analysis of clinical data did not show any difference in the behavior of tumors with p53 mutations compared with those without. Therefore, we suggest that alterations in p53 gene are an early event in the tumorigenesis of malignant osteoblastic tumors without impact on progression of these tumors.     

p53 as a sensor of carcinogenic exposures: mechanisms of p53 protein induction and lessons from p53 gene mutations

The p53 tumor suppressor gene encodes a nuclear phosphoprotein with growth inhibiting properties, which is activated in cell exposed to various forms of DNA damaging stress. The development of human cancer often involves inactivation of this suppressor through various mechanisms, including gene deletions and point mutations. Most mutations impair the specific DNA-binding capacity of p53, therefore allowing cells to proliferate in conditions where cells with intact p53 function are suppressed or eliminated. Thus, mutation of p53 may provide a selective advantage for the clonal expansion of preneoplastic or neoplastic cells. The diversity of p53 mutations provides a valuable tool to identify important sources of cancer-causing mutation in the human setting. Mutagens and carcinogens damage the genome in characteristics ways, leaving "mutagen fingerprints" in DNA. Well-characterised examples of such "fingerprints" include G: C to T: A transversions in lung cancers in association with cigarette smoke, G: C to T: A transversions at codon 249 in liver cancers in association with dietary exposure to Aflatoxin B1 (AFB1) and CC: GG to TT: AA tandem dipyrimidine transitions in skin cancers in association with UVB exposure. In addition, mutations at different codons are not functionally equivalent. The availability of crystal structures of p53 protein represents an essential development in the understanding of the functional properties of p53 mutants. In the future, it is expected that analysis of p53 mutations may provide useful information for the diagnosis, prognosis and therapy of cancer.     

Function and conformation of wild-type p53 protein are influenced by mutations in bovine leukemia virus-induced B-cell lymphosarcoma

The mutations of the p53 gene previously represented one of several genetic changes involved in the development of bovine leukemia virus (BLV)-induced lymphosarcoma, while the effects of these mutations on the function of p53 are unknown. We identified four mutations of p53 gene in BLV-infected cattle with lymphosarcoma and demonstrated clearly the existence of two functionally distinct groups of mutants: (i) the mutant forms with substitutions at codons 241 and 242, which were mapped within an evolutionally conserved region and corresponded to the human "hot-spot" mutations, had completely lost the capacities for transactivation and growth suppression and gained transdominant repression activity in p53-null SAOS-2 cells; and (ii) the mutations at codons 206 and 207 were located outside the evolutionally conserved regions. These mutants partially retained the capacity for transactivation and growth suppression and failed to inhibit the transactivation activity of coexpressed wild-type p53, instead showing an enhancement of this activity. In addition, protein analysis using an antibody specific for the mutant form revealed that the mutations at codons 206 and 242 induced a "mutant" conformation of the bovine p53 proteins. Collectively, these results show that mutations of p53 gene in BLV-infected cattle with lymphosarcoma can potentially alter its physiological function and may play an important role in BLV-induced leukemogenesis.     

Heterogeneous point mutations of the p53 gene in pulmonary fibrosis

Lung cancer is a frequent complication in pulmonary fibrosis. Overexpression of p53 proteins has been demonstrated by immunostaining in bronchoepithelial cells in patients with idiopathic pulmonary fibrosis. However, it is still unclear whether this overexpressed p53 protein is wild-type or mutant. It was hypothesized that pulmonary fibrosis may be a precancerous lesion with deoxyribonucleic acid point mutations in bronchoepithelial cells. Mutations of the p53 gene were tested for by fluorescence-based single-strand conformation polymorphism (FSSCP), cloning-sequencing and immunostaining techniques. Out of 10 tissue samples that demonstrated overexpression of p53 protein by immunostaining, nine (90%) exhibited point mutations and eight (80%) exhibited heterogeneous point mutations of the p53 gene. The mutations found in pulmonary fibrosis were scattered throughout the central part of the p53 gene, and both guanine (G):cytosine (C) to adenine (A):thymine (T) and A:T to G:C transitions were frequently observed. In conclusion, frequent heterogeneous point mutations of the p53 gene were detected in pulmonary fibrosis. These mutations may have resulted from several types of deoxyribonucleic acid damage that occurred in bronchoepithelial cells and this may explain previous findings of a very high incidence of lung cancer complicating pulmonary fibrosis.     

Overexpression of p53 protein and histologic grades of dysplasia in colorectal adenomas

PURPOSE: To clarify the relation between tumor-suppressor gene p53 expression and histologic grades of dysplasia in colorectal adenomas, we performed immunohistochemical analysis in a series of 59 colorectal polyps and 40 advanced carcinomas. METHODS: Adenomatous polyps were stained by hematoxylin and eosin and classified into mild, moderate, and severe dysplasia (intramucosal carcinoma), according to the World Health Organization's classification. RESULTS: p53 was positive in 7.1 percent (2/28) of mild, 29.4 percent (5/17) of moderate, and 62.5 percent (5/8) of severe dysplasia. In submucosal and advanced carcinomas, positivity rates were 75 percent (3/4) and 47.5 percent (19/40), respectively. Different staining patterns were found, according to grades of dysplasia. In the adenomas with mild or moderate dysplasia, a few focal crypts showed localized p53-positive staining. Adenomas with severe dysplasia had two different staining types. One was a focal staining type as shown in mild or moderate dysplasia; the other was a diffuse staining type, in which glands with mild or moderate dysplasia, surrounding severe dysplasia area, were also stained. Submucosal and advanced carcinomas showed a strong positive staining in cancer cells only. CONCLUSIONS: Overexpression of p53 protein in adenomas with mild or moderate dysplasia and existence of two types of expression in adenomas with severe dysplasia were observed. These facts suggested the possible existence of different pathways in the adenoma to carcinoma progression.