Aberrant DNA Research Articles

Simultaneous demonstration of aberrant p53 protein and HPV DNA in genital and common warts

Simultaneous demonstration of aberrant p53 protein and HPV DNA in genital and common warts

NUCLEAR-DNA CONTENT AND P53 IMMUNOSTAINING IN ORAL SQUAMOUS-CELL CARCINOMA - AN ANALYSIS OF A CONSECUTIVE 10-YEAR MATERIAL

In a retrospective analysis of 91 consecutive oral carcinomas we reviewed all patient records and weighed clinicopathological parameters against the results of image DNA cytometry and p53 immunhistochemical staining of the initial diagnostic biopsies. Eighty-seven percent of the biopsies were either aneuploid or nondiploid and there was a significant correlation between poorly differentiated tumours and aneuploidy. Sixty-nine percent of the tumours were p53 positive. DNA aberration or p53 positivity had no significant impact on prediction of survival or susceptibility to radiotherapy. In a multivariate analysis, T category had the greatest predictive value concerning survival. There was no correlation between p53 positivity and smoking.

A Monte Carlo/Markov Chain Model for the Association of Data for Chromosome Aberrations and Formation of Micronuclei

The micronucleus assay is a convenient, in situ method for observing cell damage resulting from exposure to clastogenic agents and has been widely used as a dosimeter of human exposure to radiation or chemicals. It also is a complement to the classic clonogenic cell survival assay in that it can be used to examine radiation damage vs dose as a function of cell type or radiation quality. Digitized imaging densitometry was conducted on CHO cells that have undergone one division and in which further cytokinesis was blocked to collect data on the distributions of percentage total cellular DNA per micronucleus and frequency of micronuclei per cell after gamma irradiation in G1 phase. Theoretical counterparts to both classes of distributions were generated by a Monte Carlo double-strand breakage (DSB) simulation to the CHO genome, followed by simulated repair of this initial damage using a Markov chain algorithm that assumes linear restitutions of single DSBs complete with quadratic, incomplete exchanges among pairs of DSBs. Micronuclei were presumed to consist of single acentric fragments (including fused acentric pairs). The empirical distributions, when compared to their fitted theoretical counterparts, suggest, inter alia, that: (1) a slight dependence of micronucleus size on dose exists, with a trend toward higher density in the 2-4% genome range, at the expense of the 0-2% range, with increasing dose; (2) the probability of exchange incompleteness is at least 20%; and (3) the dispersions of the micronucleus frequency distributions are progressively lower than their (essentially constant) counterparts with increasing dose. Suggested is a cooperative increase in the number of fragments per micronucleus with increasing dose. Beyond these specific results, however, it is clear that furthering the understanding of the connection between DNA aberrations and formation of micronuclei would further link these two large bodies of data.

Nuclear DNA content, proliferating-cell nuclear antigen (PCNA) and p53 immunostaining in predicting progression of laryngeal cancer in situ lesions.

Squamous epithelial cancer in situ (CIS) of the upper aerodigastric tract is a histopathologically well-defined condition. However, in clinical practice, morphological grading of dysplasia is difficult and shows large variability. The biology of CIS remains enigmatic, and there is yet no reliable way to predict whether a CIS lesion will progress to invasive cancer, remain stable or regress. In the search for markers able to foretell clinical outcome, we performed image DNA cytometry (ICM) and immunohistochemical staining for PCNA as well as p53 in 38 laryngeal CIS lesions, of which 9 progressed to invasive cancer. The majority of the CIS lesions displayed high-grade DNA aberration, a high PCNA-positive rate, and every third lesion was p53-positive by immunostaining. The lesions which progressed to invasive cancer showed a clear tendency towards more pronounced DNA aberration, a higher percentage of intense PCNA staining and more frequent p53 positivity. By combining the results from the analyses of DNA, PCNA and p53 in a prognostic index for each individual case, we correctly classified 82% of the lesions as progressors or non-progressors.

Meiotic mapping of radiation-induced DNA repair mutations and aberrations in Aspergillus nidulans: special features and use of translocations

Meiotic mapping of radiation-induced DNA repair mutations and aberrations in Aspergillus nidulans: special features and use of translocations

Proliferating cell nuclear antigen (PCNA) expression and nuclear DNA content in predicting recurrence after radiotherapy of early glottic cancer.

Proliferating cell nuclear antigen (PCNA) is a DNA replication protein maximally elevated in late G1 and S phases of the cell cycle. By using monoclonal antibodies, the expression of PCNA can be quantified and the rate of tumour cell proliferation estimated. The degree of DNA aberration in a tumour cell population reflects its genetic instability and has been implicated as a prognostic factor in an increasing number of solid tumours. The nuclear DNA content can be assessed by densitometric image cytometry DNA analysis. Both PCNA and DNA analysis can be performed on histological sections from paraffin embedded biopsies. In search of efficient and reproducible methods to identify early glottic cancers with increased risk for recurrence after radiotherapy, the PCNA expression as well as the DNA content of the diagnostic biopsies from 28 T1N0M0 glottic cancers were assessed. The group of tumours which recurred locally after radiotherapy displayed lower PCNA expression and higher DNA aberration than the group of tumours which were cured. Moreover, a combination of both parameters improved the possibility to discriminate the two groups. For T1 glottic cancer displaying high grade of genetic instability or low grade of proliferation, treatment regimes other than radiotherapy and closer follow-ups could be considered.

Aberrations of p53 gene in human hepatocellular carcinoma from China.

Allele losses and mutations have been examined in 38 cases of primary hepatocellular carcinomas (HCC) from different geographic areas of China by Southern, single-strand conformational polymorphism (SSCP) and direct DNA sequencing analyses. Two of 12 samples from Qi-Dong and six of 18 HCCs from Shanghai showed loss of heterozygosity (LOH) at the loci on chromosome 17p13.3. All of the nine mutations in the p53 gene detected in HCC from Qi-Dong were clustered at the third base of codon 249, i.e. G:C to T:A, leading to an arginine to serine change. In contrast, 18 HCC samples from Shanghai contained three mutations at codons 249, 255 and 279. These results suggested a relationship between the spectrum of p53 aberration and environmental risk factors in these two geographic areas. Since no correlation between the state of HBV DNA and p53 aberration was observed, other factors such as dietary exposure to aflatoxin B1 (AFB1) might be responsible for the mutational hotspot at codon 249 in HCCs from Qi-Dong area.

Patent Evaluation: Molecular mechanisms involved in glioblastoma formation

SummaryNovelty: The gene for human epidermal growth factor receptor (EGFR) has been shown to be altered in many glioblastomas. The aberrant DNA, RNA and protein forms of the EGFR can be used as a tumour marker, used to assay the effects of therapy or as therapy guidelines to classify tumour types.Biology: Patients with glioblastomas show amplification, overexpression and rearrangements of EGFR. An investigation using PCR to pull out the cDNA and unbalanced PCR to sequence the DNA is described here. The findings indicate that the gene for EGFR is amplified in just over 50% of tumours. The amplified gene shows structural abnormalities in 43% of the cases. Twenty-two different primers are claimed for the PCR based diagnosis of aberrant EGFR; the cDNA sequences of the aberrant EGFRs are also claimed as are monoclonal antibodies which recognize mutant EGFR proteins.

Detection of DNA aberrations in human cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products.

We have developed a simple, sensitive method, single-strand conformation polymorphism (SSCP) analysis, to detect a single nucleotide substitution in a DNA fragment amplified and labeled by the polymerase chain reaction (PCR). Mobility shift of single-stranded DNAs due to their specific conformations on non-denaturing polyacrylamide gel electrophoresis can reveal DNA aberrations. By the PCR-SSCP analysis of DNAs from surgical specimens of human cancers, mutated ras genes (17%) and aberrations of tumor suppressor p53 gene (53%) including loss of one of the two alleles and a mutation in the remaining allele were detected in lung carcinomas and aberrations of both of the p53 and retinoblastoma (RB) genes were detected exclusively in advanced hepatocellular carcinomas.

A very large proportion of human brain tumours show DNA alterations at the EGF-R gene

DNA alterations occurs frequently in sorne types of neoplasia such as retinoblastoma, and lung, breast and colorectal carcinoma, We have found that loses of restriction fragments at the epidermal growth factor receptor gene (EGEF-R) are present in as much as 82% of human brain tumours. Amplifications of fragments are present at a much lower proportion reaching only 10% of the samples examined. Two fragments of 9kb and 3.6kb are preferentially lost during tumorigenesis and the latter only is not present in 79% of the cases in which normal and tumoral samples from the same individual were compared. In all but one sample examined in which alterations are present, the 3.6kb fragment was heterozygotic in normal tissues and was not present in the tumour. Normal tissue surrounding the tumour was analyzed in a few examples and already showed a certain degree of alteration but normally not as much as the tumour itself. This will suggest that an accumulation ofseveral DNA aberrations might be responsible for determining the tumour biological propertieso Restriction fragment length polymorphism (RFLP), irrelevant to tumorigenesis, was found in about 22% of the cases.

Detection of DNA aberrations in human cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products

Detection of DNA aberrations in human cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products

Effects of an organic hydroperoxide on the activity of antioxidant enzymes in cultured mammalian cells

As a basis for an evaluation of the role of the cellular antioxidant defense system against oxidative stress, the effects of an organic hydroperoxide, tertiary-butyl hydroperoxide ( t-BuOOH), on the activity of antioxidant enzymes were investigated in cultured Chinese hamster V79 cells. Incubation of cells with t-BuOOH for 1 h significantly increased the activity of Cu-Zn superoxide dismutase (SOD) up to a level 1.4 times that of control cells. In contrast, the activities of catalase and glutathione reductase (GSSG-Rx) were not affected, while the activity of glutathione peroxidase (GSH-Px) was inhibited to a significant extent by t-BuOOH. Hydrogen peroxide also inhibited GSH-Px activity but its potency in this regard was somewhat lower than that of equimolar amount of t-BuOOH. Earlier studies demonstrated that t-BuOOH-induced cytotoxicity, single strand breaks (ssb) in DNA and structural aberrations in the chromosomes of V79 cells can be suppressed almost completely by an iron chelator o-phenanthroline. However, the iron chelator did not suppress the t-BuOOH-induced inhibition of GSH-Px activity. Likewise, a diffusible scavenger of free radicals, butylated hydroxytoluene (BHT) did not affect the hydroperoxide-induced inhibition of the enzymatic activity. These results suggest that a mechanism other than iron-mediated radical reaction is involved in the inhibition of GSH-Px activity by t-BuOOH. Modulation of the activity of antioxidant enzymes by the oxidative agent diamide was very similar to that by t-BuOOH. Inhibition of GSH-Px activity by t-BuOOH was reversible and the reduced activity returned to pre-inhibition levels within 1–2 h of post-treatment incubation. A mechanism for the inhibition of GSH-Px by t-BuOOH is discussed with reference to the oxidation of selenocysteine residues which results in perturbation of the normal catalytic cycle.

Image Cytometry DNA Analysis of Diethylnitrosamine-induce Dysplasias and Invasive Squamous Cell Carcinomas of the Esophagus in Mice

The nuclear DNA content was assessed by image cytometry in squamous epithelial dysplasias and invasive squamous cell carcinomas of the esophagus induced by diethylnitrosamine (DEN) in mice. The study comprises 48 lesions: 27 lesions with low grade (i.e. slight and moderate) dysplasia, 18 with high grade dysplasia (i.e. severe dysplasia and CIS), and 3 with invasive squamous cell carcinoma. In addition, 5 parallel run control specimens were also investigated. The results demonstrated that only 3.7% (or 1/27) low grade dysplasias but as much as 72.2% (or 13/18) high grade dysplasias, and all three invasive squamous cell carcinomas displayed non-diploid DNA values. Three of 18 high grade dysplasias and all three invasive squamous cell carcinomas demonstrated aneuploid cell nuclei. The results of the present work indicate that the esophageal mucosa of the mouse permit investigation--under controlled conditions--of the nuclear DNA alterations occurring during carcinogenesis in this organ. The results presented herein thus substantiate the theory of increasing DNA aberrations occurring during carcinogenesis in the human esophagus.

The sex-determining gene tra of Drosophila: molecular cloning and transformation studies.

In Drosophila, the primary signal for sex determination is given by the ratio of X chromosomes to sets of autosomes (X:A). The primary signal is read by a key gene (Sxl) and transmitted down to the differentiation genes by the subordinate control genes tra, tra-2, ix and dsx. Mutations in tra transform chromosomal females (X/X; tra/tra) into sterile males (pseudomales). We have cloned the tra region by microdissection and chromosomal walking. We identified the gene using deficiency breakpoints, DNA aberrations in three different alleles of tra and by P-mediated transformation. A 3.8-kb fragment perfectly rescued the mutant phenotype of X/X; tra/tra flies, showing that it contained all the necessary information to restore female-specific functions in the mutant flies. We present evidence that most of the function of tra can be provided by a subsegment of 2 kb that is differentially transcribed or processed in males and females.

Relation between flow cytometric DNA distribution and pathology in human bladder cancer. A report on 69 cases.

High-resolution flow cytometric measurements of cellular DNA content have been performed in 69 patients with transitional cell carcinomas of the urinary bladder. By selective sampling of cells at cystoscopy, mapping of the whole bladder could easily be performed, including the tumor as well as different areas of the surrounding normal-appearing mucosa. The cell-cycle distribution showed an increasing fraction of cells with S- and G2-phase content parallel to the World Health Organization (WHO) grade of the tumors, ranging from 1.7% S-phase cells in normal subjects to nearly 20% in tumors of WHO grade 3. WHO grade 2 tumors could be divided into two populations: (1) diploid, with a cell-cycle distribution similar to grade 1 tumors and low frequency of infiltration; and (2) aneuploid, with a high rate of infiltration and high fractions of S- and G2-phase cells. Furthermore, the aneuploid tumors could be subdivided into two main classes, one hypertriploid with high frequency of involvement of the surrounding normal appearing mucosa; and one hypotetraploid, with less frequency of such involvement and infiltration. It is concluded that by selective sampling and mapping of the bladder mucosa using flow cytometry, subclassification of this type of tumor can be performed rapidly, giving a reliable measure of the involvement of the bladder in the neoplastic disease and a biologic subclassification based on the type of DNA aberrations.

DNA aberrations of bladder mucosa in patients with transitional cell carcinomas.

DNA aberrations in bladder mucosa have been investigated in altogether 26 patients with aneuploid WHO grade 2 and 3 tumours (transitional cell carcinomas; TCC). In about 1/4 of the patients aneuploid cells were found only in the tumour. Hypotetraploid tumours showed the lowest frequency of involvement of the normal appearing mucosa of the trigone (43%). In hypertriploid tumours the corresponding value was 71%. In altogether 14 of the patients, the same type of aneuploid cells was found in the normal appearing mucosa as in the tumour. This indicates frequent involvement of the whole bladder mucosa in the tumour disease. In about 1/4 of the cases other types of ploidy aberrations were found in the normal appearing mucosa than in the tumour. This is indicative of preneoplastic changes in the mucosa, of which only one type of aberrations is associated with tumour growth. In some cases with multiple tumours of the bladder, all the tumours had the same aneuploid stemline, while in other cases the tumours were of different aneuploid stemlines. This is conformal with other reports of atypia in the surrounding mucosa in TCC and with the concept that recurrence of high grade tumours is the consequence of neoplastic involvement of the whole bladder.

Ataxia-telangiectasia: A multiparameter analysis of eight families

Eight families, including 12 patients with ataxia-telangiectasia (AT), were investigated for parameters which tested the current models of pathogenesis. We found: T cells decreased, B cells elevated, PHA response decreased, variable T suppressor activity, serum IgA and IgG 2 decreased. AFP and liver enzymes in serum were elevated. In addition, several parameters related to cell membrane and cytoskeletal functions were abnormal: increased concanavalin A capping and increased cyclic nucleotides of lymphocytes and decreased chemotactic responses of neutrophils. Thymosin-α-1 levels of patients and family members did not differ from age-matched controls. While many of the immunological abnormalities seen in AT could be due to a common immunoregulatory defect, this would not explain the neurological deterioration or the DNA aberrations. We suggest that a broader mechanism be considered in future analyses, such as the possibility that a primary or secondary cytoskeletal disorder might underlie the myriad abnormalities seen in AT.

Clinical application of flow cytometry: A review

A survey of the use of flow cytometry for clinical purposes is given. In the last decade the main clinical application of this technique has been measurements of cellular DNA content for estimation of cell cycle distribution and ploidy studies. A large body of data is now available on the presence of aneuploidy in different malignant diseases. By measurements with high resolution, the demonstration of abnormal cellular DNA content in several types of tumors can be of definite diagnostic value when combined with conventional diagnostic procedures. The prognostic significance of different types of DNA aberrations is so far not established. Attempts to monitor cancer treatment by studying altered cell cycle distribution have not been successful, although some applications are of potential value. The main reasons for this are the complexity of tumor tissue as well as difficulties with interpretation of altered cell cycle distribution caused by drug combinations. For further progress in this field more emphasis on other cell constituents than DNA measured by flow cytometry is desirable, either as single or as multiparameter measurements.

Genetic and cytological characterisation of fusion chromosomes of Dictyostelium discoideum.

Abnormally large chromosomes which appear to result from the fusion of 2 chromosomes of the normal karyotype have been found in diploids of Dictyostelium discoideum formed by parasexual fusion of haploid strains HU483 (N = 7) and HU245 (n = 7). These fusion chromosomes appear to be the products of the tandem translocation of most, if not all, of one acrocentric chromosome to the telomere of a second acrocentric. Thus the chromosome number of the diploids is reduced from the normal 2n = 14 to 2n = 13 with the formation of an abnormally large acrocentric fusion chromosome. Experimental haploidisation of such diploids result in two types of products, those with a normal 7 chromosome karyotype and those with an abnormal 6 chromosome karyotype which contains the fusion chromosome. Genetic analysis of haploid segregants indicates that linkage groups II and VII are involved in this fusion. Phenotypes of recombinant diploids obtained following mitotic crossing-over establishes that linkage group II is proximal to linkage group VII. Cytological examination of the karyotypes of haploid strains bearing the fusion chromosome suggest that chromosome 2 may correspond to linkage group II and chromosome 3 to linkage group VII. Haploid strains bearing the fusion chromosome grow and develop normally so little or no genetic information can have been lost in the fusion event. While the nature of this event is unknown it may have involved aberrant recombinational DNA repair since the parental haploid strain HU483 bears the radB13 DNA repair mutation.

A mutant immunoglobulin light chain is formed by aberrant DNA- and RNA-splicing events.

A mutant immunoglobulin gene has been formed by an abnormal (non V/J) recombination event such that abnormal RNA splicing is required to form a mutant light chain. The structure of the gene suggests that the small palindrome thought to be involved in V/J joining also provides the basis for this abnormal DNA recombination and that the absence of a J segment and RNA splice signal allows an abnormal RNA splicing reaction to occur.