Effect of oxidative insult on young and adult cardiac muscle cells in vitro.

The effect of hydrogen peroxide on cultured neonatal and adult cardiac myocytes was investigated. On neonatal cardiac myocytes the effect was very pronounced at a low concentration (0.03 mM), whereas the adult cardiac myocytes were resistant to the same concentration of H2O2. Dividing neonatal cardiac myocytes were more susceptible to H2O2 insult than the non-dividing adult cardiac myocytes. At a concentration of 0.1 mM H2O2, the neonatal cardiac myocytes were significantly damaged compared with the adult cardiac myocytes. Cardiac muscle cells from neonatal and adult hearts at high density culture were more tolerant to the oxidative insult by H2O2 than cells in low density culture. The damaging effect of H2O2 was very selective on F-actin in neonatal and adult cardiac muscle cells. The effect of H2O2 on myosin, titin, alpha-actinin, desmin or tubulin was not pronounced. Microscopical studies suggested a more marked protection by catalase than by glutathione reductase in the neonatal cells.

A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue.

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.

Analysis of p53 gene mutations in human gliomas by polymerase chain reaction-based single-strand conformation polymorphism and DNA sequencing.

Mutations in the p53 gene have been recognized in brain tumors, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for frozen tissues. We have developed a method utilizing polymerase chain reaction (PCR) for the direct analysis of p53 mutation by single-strand conformation polymorphism (SSCP) and by direct DNA sequencing of the p53 gene using a single 10-microns paraffin-embedded tissue section. We applied this method to screen for p53 gene mutations in exons 5-8 in human gliomas utilizing paraffin-embedded tissues. Twenty paraffin blocks containing tumor were selected from surgical specimens from 17 different adult patients. Tumors included six anaplastic astrocytomas (AAs), nine glioblastomas (GBs), and two mixed malignant gliomas (MMGs). The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure > 90% of the tumor cell population for p53 mutational analysis. Simultaneously, microdissection of the tissue was also carried out to obtain normal tissue from adjacent areas as a control. Mutations in the p53 gene were identified in 3 of 17 (18%) patients by PCR-SSCP analysis and subsequently confirmed by PCR-based DNA sequencing. Mutations in exon 5 resulting in amino acid substitution were found in one thalamic AA (codon 158, CGC > CTT: Arg > Leu) and one cerebral hemispheric GB (codon 151, CCG > CTG: Pro > Leu).(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of human papillomavirus (HPV) DNA in human prostatic tissues by polymerase chain reaction (PCR).

Human papillomavirus (HPV) infections are strongly linked to the pathogenesis of uterine cervical neoplasms, and have been implicated in other cancers of the female genital tract. In contrast, the association of HPV with the cancers of the male urogenital tract is less evident, except in anal and penile cancers. However, recent studies reporting the prevalence of HPV infections in human prostate cancers (60-100% HPV 16 positive vs. no infection of HPV) have raised controversies regarding the prevalence of HPV in benign and neoplastic human prostate. We investigated the prevalence of HPV infections in prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinomas in 23 surgically resected prostates. Polymerase chain reaction (PCR) was used to amplify HPV 6b/11, 16, and 18 specific DNA sequences, using type specific HPV primers selected from the transforming gene E6-E7. The areas of PIN and cancer in 6 microns H&E stained tissue sections were identified, and respective areas of PIN and cancer were isolated from the adjacent serial sections and used for DNA amplification and HPV detection (Fig. 1). Our results demonstrated the presence of HPV 16 in three carcinomas (13%), using type specific primers in PCR amplified samples. We were not able to demonstrate the presence of other HPV types (HPV 6b/11 or HPV 18) in any of the samples using specific primers. Two of these prostates showed relatively strong positive signals by dot blot analysis, when hybridized with a 32P-labeled HPV 16 type specific oligonucleotide probe. One more sample showed weak positivity, when hybridized with a 32P-labeled HPV 16 type specific oligonucleotide probe. Subsequently, we have confirmed these results by Southern hybridization of the samples transferred to nylon membrane after agarose gel electrophoresis and detected by HPV 16 type specific oligonucleotide probe, using chemiluminescent assay. We, therefore, conclude that HPV infections of the prostate in general are not as common as has been previously claimed by other investigators.

Measurement of cellular proliferation in human prostate by AgNOR, PCNA, and SPF.

Tumor differentiation and proliferative activity are important predictors of biological behavior. While routine histological evaluation is fairly adequate to assess differentiation, tumor proliferative activity is difficult to measure. Silver staining for nucleolar organizer regions (AgNORs) is reported to be helpful for assessing tumor proliferation. We investigated the AgNOR counts in 20 formalin fixed, paraffin embedded human prostate tissues in three microscopic fields of 330X, using an image analysis system. A total of 200-700 nuclei were evaluated on histologically controlled areas of nonneoplastic prostate tissue, prostatic intraepithelial neoplasia (PIN), and invasive carcinoma. The values were compared to flow cytometrically obtained synthesis phase fractions (SPF) and immunohistochemically semi-quantitated, proliferative cell nuclear antigen (PCNA) patterns. AgNOR counts were also compared to tumor stage and Gleason's score. The pattern of PCNA staining in formalin fixed specimens was widely variable, probably due to differences in preservation of antigen. The positive counts varied from 0 to 55%, with a mean value of 8.55 +/- 15.9. The SPF values ranged from 5 to 13% with a mean value of 8.50 +/- 2.37. Two of 20 tumors were aneuploid and 18 were of diploid range. The mean AgNOR values in nonneoplastic nuclei (1.836 +/- 0.299), PIN (3.129 +/- 0.295), and invasive tumor cell nuclei (4.737 +/- 0.369) were highly significant (P < 0.0001) when paired differences were compared. AgNOR counts correlated significantly with tumor Gleason's score (P < 0.0145). However, the correlation coefficient for SPF and AgNOR values was not significant (P > 0.24), possibly because of the small number of samples examined. The highest AgNOR counts were found in the two aneuploid tumors. We conclude that AgNOR count may be a potential indicator of cellular proliferation, and possibly a marker of tumor differentiation.