Lysyl oxidase activity in the cells of flexor retinaculum of individuals with carpal tunnel syndrome.

Lysyl oxidase (LO) is produced by myofibroblast cells in some tissues and can be influenced by transforming growth factor beta 1 (TGF beta 1). Myofibroblast-like cells are present in the flexor reticulum of patients with carpal tunnel syndrome (CTS). The goal of the current study was to determine LO activity and the effects of TGF beta 1 on LO expression in the cells from patients with CTS. Tissues from both hands of five individuals with CTS were used for this study. LO activity with and without TGF beta 1 stimulation was assayed in 7-day cell culture specimens. A significant difference in LO activity among individual patients, but not between right and left hands of the same patient, was observed. There was no correlation between the severity of CTS determined by nerve conduction studies and LO activity. Addition of TGF beta 1 significantly increased LO in all cell lines.

Explant culture, immunofluorescence and electron-microscopic study of flexor retinaculum in carpal tunnel syndrome.

Although flexor-retinaculum (FR) release provides dramatic relief from carpal tunnel syndrome (CTS), the role of this ligament in CTS is not well understood. We have adopted a unique approach to study the cellular pathogenesis of CTS by establishing a method for the culture of cells of FR from subjects with and without CTS. The cultured cells were characterized by light, immunofluorescence, electron microscopy, Western blot analysis, and growth studies. Two main differences between the CTS and control cells included a faster growth rate and an altered fine morphology that reveals the contractile nature of the CTS cells. It is possible that the presence of these contractile cells in FR is responsible for increasing the contractility of the FR, leading to a decrease in the volume of the carpal tunnel, thus exerting pressure on the median nerve and triggering CTS.

Differential effects of nitrogen mustard in vivo on the cancer and host cells in MCa-11 tumours.

We analysed the effects of nitrogen mustard (HN2) on the growth, cell cycle distributions, and ratios of tumour cells to host cells for MCa-11 tumours grown in vivo. Treatment of tumour-bearing BALB/c mice with 3 mg/kg of HN2 produced a significant slowing of MCa-11 tumour growth. Seventy-two hours after treatment in vivo with either 3 or 4 mg/kg of HN2, the host cells in the treated tumours showed a significantly decreased G0/G1 peak and an increased G2/M peak (P < 0.01), whereas the cancer cells in the treated tumours showed significant increases in the G0/G1 peak coupled with relatively decreased proportions of S and G2/M tumour cells (P < 0.001). The ratio of the total number of cancer cells to the total number of host cells in the tumours was significantly increased 72 h after HN2 administration (P < 0.01). Thirty-two days after treatment with HN2, the cell cycle distributions of the host and tumour cells in the treatment and control tumours had returned to being identical, but the ratio of the total number of cancer cells to the total number of host cells remained increased in the treated tumours (P < 0.01). These results show that the administration in vivo of HN2 can lead to entirely different cell cycle effects for the host and cancer cells in the same tumour, and that the partial growth arrest of MCa-11 tumours from HN2 treatment may be due in part to the preferential destruction of host cells rather than solely to a direct cytotoxic effect on the cancer cells.

K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.

We examined 82 surgically resected or biopsied, formalin-fixed, paraffin-embedded primary adenocarcinomas of the pancreas for the presence of activating point mutations in codon 12 of the K-ras oncogene. Mutations were detected using primer-mediated, mutant-enriched, polymerase chain reaction-restriction fragment length polymorphism analysis and characterized further by allele-specific oligonucleotide hybridization. This combination of mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis and allele-specific oligonucleotide hybridization results in a rapid and sensitive characterization of the mutations in codon 12 of K-ras. Sixty-eight (83%) of the 82 carcinomas examined harbored a point mutation. Of the 68 mutations, 33 (49%) were guanine to adenine transitions, 27 (39%) were guanine to thymine transversions, and eight (12%) were guanine to cytosine transversions. Mutations were found in carcinomas of the head (61 of 75, 81%) as well as in carcinomas of the body or tail (seven of seven, 100%) of the pancreas. The overall prevalence of K-ras point mutations in adenocarcinomas of the pancreas obtained from patients who smoked cigarettes at some point during their lives (88%; 86% in current smokers and 89% in ex-smokers) was greater than that seen in pancreatic adenocarcinomas from patients who never smoked cigarettes (68%, P = 0.046). The presence of K-ras point mutations did not correlate with tumor ploidy, tumor proliferating index, or patient survival. These results demonstrate that primer-mediated, mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis combined with allele-specific oligonucleotide hybridization can be used to detect and characterize mutations in codon 12 of the K-ras oncogene in formalin-fixed, paraffin-embedded tissues, and the results confirm that activating point mutations in codon 12 of the K-ras oncogene occur frequently in adenocarcinomas of the pancreas.

A comparison of flow cytometric and absorption cytometric DNA values as prognostic indicators for pancreatic carcinoma.

BACKGROUND: The DNA content of 30 adenocarcinomas of the head of the pancreas was measured by flow and absorption cytometric analysis. METHODS: Each of the patients in this study had curative pancreatoduodenectomy. The absorption cytometric measurements were done in a research laboratory, and the flow cytometric measurements were performed in a commercial laboratory. The DNA measurements were done on nuclei disaggregated from pancreatic cancer tissue blocks without the examiner knowing whether the patient had survived. RESULTS: Twenty-one of the 30 cancers were found to be aneuploid by absorption cytometric analysis, whereas only 1 of the 30 cancers was aneuploid by flow cytometric analysis. This difference was statistically significant (P < 0.001). CONCLUSIONS: Univariate and multivariate analyses showed that the absorption cytometric DNA measurements were stronger prognostic determinants for patient survival than were the flow cytometric DNA measurements, indicating that some caution may be warranted in the interpretation of commercially obtained DNA distributions of pancreatic carcinomas.

Pancreatic cancer cell DNA content correlates with long-term survival after pancreatoduodenectomy.

The DNA content of 47 adenocarcinomas arising in the head of the pancreas from patients who had undergone successful pancreatoduodenectomy was measured. The DNA measurements of each tumor were made without knowledge of the clinical course by absorption cytometry performed on Feulgen-stained nuclei that had been disaggregated from pancreatic cancer tissue blocks. Forty-seven evaluable DNA distributions were obtained from specimens taken between 1975 and 1988. Of the 47 tumors, 19 (40%) were diploid and 28 (60%) were aneuploid cancers. The 19 patients with diploid cancers had a median survival time of 25 months. Median survival of the 28 patients with aneuploid cancers was 10.5 months. This difference was statistically significant (p = 0.003). A multivariate life table regression analysis demonstrated that the ploidy and proliferative index as determined by absorption cytometry were independent prognostic factors, as strong as or stronger than the number of positive nodes and tumor size. Thus cellular DNA content appears to be one of the most important predictors of survival in patients with adenocarcinoma of the head of the pancreas who have successfully undergone a pancreaticoduodenectomy.

Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells.

We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obtained by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.

Slowing of cell cycle traverse for cells in exponential monolayer cultures placed into plateau-fed and starved medium.

MCa-11 tumor cells in exponential monolayer cultures were pulse/chase-labeled with [3H]thymidine and then regrown in fresh, plateau-fed, or starved medium. We measured the DNA content and autoradiographic labeling of these cells by absorption cytophotometry at intervals of 0, 2, 4, 8, 12, and 24 h to follow the progress through the cell cycle of those cells which had incorporated isotope. We found that for the cells grown in plateau-fed and starved medium the G0/G1, S, and G2 phases of the cell cycle were prolonged when compared to those for cells grown in fresh medium. These results show that, under adverse microenvironmental conditions, the growth of tumor cells can be regulated in all phases of the cell cycle, and that this regulation can include lengthening and even cessation of replicative DNA synthesis.

The cytogenetic and hepatotoxic effects of dioxin on mouse liver cells.

The cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on mouse liver cells were investigated. Male C57BL/6J strain mice, which have TCDD receptors, were given single intraperitoneal injections of 25, 37.5, 75 and 150 micrograms of TCDD/kg body weight or corn oil carrier alone. Two-thirds hepatectomies were carried out at 1 or 7 days after injection and chromosomal aberrations and mitotic indexes of the regenerating hepatocytes were scored 54 hr after hepatectomy. Liver sections from additional intact mice were studied for TCDD-hepatotoxicity at 1, 7 and 30 days after injection. The three high doses of TCDD caused hepatotoxicity with necrosis of liver cells and focal architectural collapse by 30 days after injection. No evidence was obtained of an increase in the frequency of chromosomal structural aberrations at doses that allowed sufficient mitotic activity for cytogenetic evaluation. We conclude that TCDD is not a clastogen for mouse hepatocytes, although high doses cause marked hepatocellular necrosis.

Cytophotometric determination of unscheduled DNA synthesis.

We describe a cytophotometric assay for unscheduled DNA synthesis (UDS) in asynchronously growing cells. Monolayer cultures of human HEp-2 and mouse MCa-11 cells were incubated with the carcinogen methyl-methane sulfonate (MMS), as well as with hydroxyurea and (3H)thymidine. Slides were prepared, and the DNA contents and areas of nuclei were measured by absorption cytophotometry. The labeling of the nuclei, determined on the basis of their DNA content to be in G0/G1, was selectively measured after the preparation of autoradiographs. The labeling of the G0/G1 cells increased with increasing doses of MMS. We also found that the increased nuclear labeling after MMS treatment was not due to induction of replicative DNA synthesis or selective destruction of G0/G1 cells. The results of this assay compared favorably with a standard biochemical method for measuring unscheduled DNA synthesis by benzoylated naphthoylated DEAE cellulose chromatography.

Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases.

In vitro DNA synthesis on a phi X174 template primed with a restriction fragment and catalyzed by the Escherichia coli DNA polymerase I large (Klenow) fragment (pol I) terminates at the nucleotide preceding a site that has been altered by ultraviolet irradiation or treatment with N-acetylaminofluorene. Termination on ultraviolet-irradiated templates is similar when synthesis is catalyzed by E. coli DNA polymerase III holoenzyme (pol III), phage T4 DNA polymerase, a polymerase alpha from human lymphoma cells, or avian myeloblastosis virus reverse transcriptase. 3' leads to 5' exonuclease activity cannot be detected in the reverse transcriptase and DNA polymerase alpha preparations. On N-acetylaminofluorene templates, pol I, pol III, and T4 polymerase reactions terminate immediately preceding the lesion, whereas reverse transcriptase-catalyzed reactions and, at some positions in the sequence, polymerase alpha-catalyzed reactions terminate at the site of the lesion. Substitution of Mn2+ for Mg2+ changes the pattern of pol I-catalyzed termination sites. The data suggest that termination is a complicated process that does not depend exclusively on the 3' leads to 5' exonuclease activity associated with many polymerases.

Apurinic/apyrimidinic endonuclease sensitive sites as intermediates in the in vitro degradation of deoxyribonucleic acid by neocarzinostatin.

Neocarzinostatin (NCS) induces alkali-labile sites in DNA which are stabilized by NaBH4 reduction. The stabilized sites are sensitive to an AP endonuclease from human lymphoma cells. NCS-induced degradation of supercoiled Col E1 DNA proceeds in stepwise fashion with apurinic/apyrimidinic (AP) sites as intermediates. Degradation is increased when reaction occurs in the presence of AP endonuclease, and DNA reacted with NCS can be shown to have numerous AP endonuclease sensitive sites.

Repair of neocarzinostatin-induced deoxyribonucleic acid damage in human lymphoblastoid cells: possible involvement of apurinic/apyrimidinic sites as intermediates.

Neocarzinostatin (NCS) induces repair in a xeroderma pigmentosum lymphoblastoid line deficient in the ability to repair DNA damage induced with (acetoxyacetyl-amino)fluorene. Repair was demonstrated by the induction of repair synthesis and by the disappearance of NCS-induced single-strand breaks and/or alkaline-labile sites in DNA. Estimation of NCS-induced repair patch size, based on the density shift induced in DNA by extensive shear after incubation of treated cells in medium with bromodeoxyuridine or by calculation from the extent of restoration of DNA sedimentation profiles in alkaline sucrose gradients and the amount of repair synthesis measured by the BND cellulose method, indicated that only a few nucleotides were inserted per repaired region. NCS-treated bacteriophage T7 DNA requires incubation with alkaline phosphatase to make it a substrate for DNA polymerase I. NCS-reacted T7 DNA, even after phosphatase treatment, is not a substrate for a DNA polymerase alpha obtained from human lymphoma cells. NCS-treated T7 DNA did serve as a substrate for the DNA polymerase alpha when incubated with an apurinic/apyrimidinic (AP) endonuclease with associated 5'-3'-exonuclease activity. The results suggest that NCS-induced AP sites could be intermediates for the in vivo repair synthesis.

Specific binding of Excherichia coli chain Initiation factor 2 to fMet-tRnafMet.

A stable Escherichia coli IF-2-fMet-tRNAfMet complex is formed upon incubation of IF-2 (prokaryotic initiation factor) with fMet-tRNAfMet is the presence of 50 mM Tris-HCl, pH 7.0, 100 mM NH4Cl, and 7 mM 2-mercaptoethanol. The complex thus formed is retained on a Millipore filter and is assayed accordingly. Complex formation does dot require GTP, is unstable in the presence of 5 mM Mg2+, and is specific for fMet-tRNAfMet. Other amino acyl-tRNAs or deacylated tRNAs do not form such a complex with IF-2. A crude ribosomal high salt wash preparation contains other protein factors which bind unspecifically to RNAs under the above binding conditions. One of these factors elute similarly to IF-1 on DEAE-cellulose chromatography. Extensively purified IF-1 and IF-3 show weak and unspecific RNA-binding activities. The RNA-protein complex formed in each of the above cases, like the IF-2-fMet-tRNAfMet complex, is retained on Millipore filter and is sensitive to Mg2+.