Cellular F-actin levels as a marker for cellular transformation: relationship to cell division and differentiation.

Transformation is associated with profound structural and quantitative changes in the cytoskeleton. Herein we report studies using F-actin, a major cytoskeletal protein, as a quantitative marker for transformation cells, focusing on separating the effects of the cell cycle, cell differentiation, and transformation. The model system for these studies consisted of three lymphoblastic cell lines, one untransformed line (RPMI) and two transformed lines, one (HL-60) of which can be induced to differentiate and the other (Daudi) which cannot. The relation of F-actin levels to cell cycle was studied by flow cytometry with the use of fluorescein-phalloidin to label F-actin and propidium iodide to label DNA. F-actin levels in transformed Daudi and HL-60 lines were only two-thirds that of the untransformed RPMI cells. Histograms of the distribution of F-actin showed that the transformed lines consisted of two cell populations, one having an F-actin content near that of untransformed cells and the other having much less. Cell cycle analysis showed that F-actin in untransformed cells increased 10-15% as cells entered the S compartment, remaining approximately constant through G2 + M phases of the cell cycle, but in transformed cells the major increase in F-actin occurred during G2 + M phase. Double-label studies with rhodamine-phalloidin for F-actin and KI-67 monoclonal antibody for dividing cells (cells at late G1, S, G2, and M) measured with quantitative fluorescence image analysis showed that the mean F-actin content of dividing cells was twice that of nondividing cells. These results suggested that most of the cell division-related F-actin increase occurred during late G1 phase in untransformed cells. Differentiation of HL-60 cells with dimethyl sulfoxide or retinoic acid normalized the F-actin content of the nondividing cell population, but dimethyl sulfoxide and retinoic acid produced no detectable change in F-actin in the undifferentiable Daudi cells. A tumor promoter (12-O-tetradecanoylphorphol-13-acetate) inhibits differentiation of hematopoietic cells, resulted in a 32% decrease in the mean F-actin content of RPMI cells due to the appearance of a new subpopulation of low F-actin content. The 12-O-tetradecanoylphorbol-13-acetate-induced changes reversed slowly after removal of 12-O-tetradecanolyphorbol-13-acetate but more rapidly in the presence of retinoic acid. These results indicate that F-actin quantification can serve as a marker for cellular transformation and provides a tool for studying the mechanisms of cellular differentiation that may lead to a better understanding of the oncogenic process.

Equilibrium binding of Hoechst 33258 and Hoechst 33342 fluorochromes with rat colorectal cells.

We examined the biophysical characteristics of the interaction of Hoechst 33258 and 33342 dyes with normal rat colorectal cells as functions of fixation and solution composition. Classical dye-binding techniques were used to investigate the stoichiometry and binding constants with whole cells, and quantitative fluorescence image analysis was used to specifically study nuclear dye binding in intact cells. In aqueous solution, H-33258 dye bound cooperatively with intact cells, with a binding constant of between 3-4 x 10(5). In ethanolic solution, binding appeared less cooperative, although Scatchard analysis could not be used. The binding constant was slightly lower (2 x 10(5)), but the total number of cell binding sites was decreased by a factor of 5, reflecting a great decrease in cytoplasmic sites. QFIA studies identified conditions optimal for DNA quantitation under which the fluorescence signal was independent of dye or cell concentration. The proportionality between absolute nuclear fluorescence intensity and DNA content was established, and the upper limit of DNA content of normal colorectal cells was also determined.

Early detection of colorectal cancer by quantitative fluorescence image analysis of exfoliated cells.

Early-stage colorectal cancer is potentially curable. In the present study, we applied quantitative fluorescence image analysis (QFIA) cytology to the detection of experimental colorectal cancer in a rodent model. QFIA cytology combines visual cytologic examination with quantitation of DNA content in single exfoliated cells. Cancer was induced by treating 110 rats with subcutaneous 1,2-dimethylhydrazine. Sequential colon washes were obtained weekly from each animal for 20 weeks. Control animals were treated identically except for the administration of carcinogen. Cells that were cytologically abnormal or had increased DNA content were found starting in the second week. By the eighth week, roughly 50 percent of animals had positive results, and this level remained approximately constant for the duration of the study. Tissue pathologic results were normal during weeks 1 to 7. Dysplasias became common during weeks 8 to 15 whereas most cancers appeared during weeks 16 to 21. These results indicate that QFIA cytology is a highly sensitive method for detecting even preneoplastic changes resulting from carcinogen administration and may prove useful in detecting human colorectal cancer.

Family care in a family practice group.

A fundamental claim of family medicine is that the family physician treats the "whole" family--an ideological principle that guides undergraduate and residency education. Using archival data obtained from a random sample of 500 patients in one group of family physicians, this study analyzed the extent to which this principle is carried out in practice. Physicians trained in family practice residency programs were compared with their colleagues. Family types and marital stability were also examined. Results indicate that in only 28 percent of families (excluding single person households) were all family members seen by the same family physician. This occurred despite the high preponderance and stability of traditional nuclear families in the practice. There was no significant difference in this rate between graduates and nongraduates of approved family practice residency programs.

Routine neonatal circumcision: the gap between contemporary policy and practice.

Attitudes, knowledge, and personal factors related to circumcision in the newborn period were analyzed among a group of 92 randomly selected primary care physicians and 103 parents of male infants. Sixty-five percent of the physicians conveyed a positive attitude about routine neonatal circumcision to their patients; pediatricians were more likely to have a neutral attitude, and both family and general practitioners were more likely to encourage routine neonatal circumcision (P less than .01). Routine neonatal circumcision was favored more often by older, male, and circumcised physicians. Knowledge about the normal anatomy of the infants' foreskin was inadequate. Parents rarely perceived physicians as influential in the decision-making process (P less than .001). In contrast, fathers' circumcision status and parental belief in medical indications were positively related to the decision to circumcise (P less than .001 and P less than .01, respectively). The data suggest directions for change in clinical pediatric practice that may bring contemporary policy with regard to routine neonatal circumcision closer to actual practice.

Breast self-examination in the primary care setting.

Very little is known about breast self-examination (BSE), a health practice that has been found to be associated with better clinical and pathological breast cancer outcomes. Using data obtained from a sample of 260 women frequenting three primary care centers, this study investigates rates of BSE and how such rates are affected by social and medical factors. Results indicate that BSE practices are influenced to some extent by a woman's race and level of education, while they are significantly affected by her religion. In contrast, there was no evidence to suggest that a woman's knowledge of breast cancer risk factors, or her own level of risk for the disease, affect the extent to which she is likely to self-examine. These findings suggest the need to confirm these social influences on BSE practice using a larger, population based sample. Moreover, the values and attitudes affecting differential rates of BSE performance should be further investigated.

Response of physicians to medical complaints in men and women.

Workups by physicians in response to five common complaints in a sample of 104 men and women--52 married couples--were evaluated by chart audit. For the total group of complaints, back pain, headache, dizziness, chest pain, and fatigue, the physicians' workups were significantly more extensive for men than they were for women. These data tend to support the argument that male physicians take medical illness more seriously in men than in women.

The effect of a surface disinfectant on a dental cast.

This study determined the effect of a disinfectant solution on dental casts. Stone samples were immersed in a disinfectant solution and in control solutions. The results indicate that a saturated calcium sulfate (clear slurry) solution with 0.525% sodium hypochlorite was an effective disinfectant and acted without damage to the dental cast.

Quantitative fluorescence image analysis in bladder cancer screening.

Quantitative fluorescence image analysis (QFIA) cytology combines image analysis to measure DNA with visual cytology for bladder cancer detection. QFIA sensitivity is 76% to 81% and 95% to 100% for low- and high-grade tumors respectively, with 94% specificity in asymptomatic controls. QFIA screening of 504 persons within a beta-naphthylamine exposed cohort found DNA hyperploidy correlated with the duration of carcinogenic exposure and smoking history; marker prevalence was 23% for exposed workers who smoked and 2% for nonexposed nonsmokers. In prospective animal studies, QFIA was useful in monitoring carcinogenesis and chemoprevention with retinoids. Current QFIA research focuses on measurements of oncogenes, growth factors and their receptors, cytoskeleton, and tumor-associated antigens to improve sensitivity and specificity to low-grade tumors and to identify premalignant conditions. Profiles of biochemical and immunological markers on single cells may further assist in the study of high-risk cohorts and individual risk assessment.

Ethnic group response to preventive health education.

Using the ratio of preventive visits to episodic visits as an index of health care utilization in response to an education campaign which emphasized the value of prevention, we analyzed the response of white, Mexican-American, and black subjects in a community health center. We found that: (1) whites make a relatively larger proportion of preventive care visits than Mexican-Americans, who made proportionately more preventive health care visits than blacks. The difference between whites and blacks was statistically significant (p less than .05). (2) When socioeconomic levels were held constant, as measured by insurance categories, no ethnic differences in preventive vs episodic health care visits were found. (3) On the other hand significant differences in such health care behavior emerged when indices of family structure and employment were isolated. Mexican-Americans seek proportionately less preventive care in families in which one parent is absent and no adult is employed. In contrast, blacks seek proportionately less preventive care in families in which both parents are present at at least one adult is employed.

DNA cytometry and cytology by quantitative fluorescence image analysis in symptomatic bladder cancer patients.

A semi-automated quantitative fluorescence image analysis (QFIA) technique was developed with the Leitz TAS-Plus to detect bladder cancer using hyperploidy in urinary cells. Absolute nuclear fluorescence intensity (ANFI) (emission at 540 nm with excitation at 436 nm) of individual acridine-orange-stained cells was quantitated using (1) QFIA and (2) simple filter microspectrofluorophotometry (SFM). Both methods employed an internal phosphor particle standard which, when once calibrated against the DNA content of normal cells, obviates the necessity of routinely calibrating against normal cells in each sample. Results of SFM and QFIA were compared with routine Papanicolaou (Pap) cytopathology, using histopathology as the diagnostic standard in 272 samples from 67 symptomatic patients. The sensitivities for detecting low-grade transitional-cell carcinoma were 86% for SFM, 76% for QFIA, and 33% for Pap cytology. QFIA and SFM were significantly more sensitive at detecting bladder cancer than was Pap (0.01 greater than p greater than 0.001). Comparison of sensitivity obtained with bladder washings and urine samples showed that noninvasively obtained urines can be used. ANFI also detected recurrent and precancerous bladder lesions and kidney, ureter, and prostate lesions. This approach may prove generally useful in quantifying biochemical and immunological probes and should be broadly applicable as a research tool for studying the relationship of biochemical markers in the pathogenesis of disease and as a test for cancer control.

Quantitative fluorescence image analysis of deoxyribonucleic acid ploidy in urine from normal children.

Quantitative fluorescence image analysis incorporates the 2 diagnostic techniques of cytological analysis with quantitation of deoxyribonucleic acid (DNA). Exfoliated urinary cells are ideal for analysis by this method, which allows the identification of "rare event" abnormal cells. We evaluated the urine from 50 children who had undergone cystoscopy or were catheterized for other reasons. The urine was free of infection by urinalysis. Cytological analysis demonstrated normal or atypical cells in all patients. Of the patients 1 (2%) had greater than 2 of 500 cells analyzed with greater than 5C DNA and 4 (8%) had greater than 2 of 500 cells with greater than 5C double stranded nucleic acid. These data suggest that it may be "normal" for urine to contain "rare event" abnormal cells. The significance of this finding is unclear at present.

Nonribosomal peptide synthesis and toxigenicity of cyanobacteria.

Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate function of large, modular enzyme complexes known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial biosynthesis (R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, and C. Khosla, Science 262:1546-1557, 1993; T. Stachelhaus, A. Schneider, and M. A. Marahiel, Science 269:69-72, 1995). Most nonribosomal peptides from microorganisms are classified as secondary metabolites; that is, they rarely have a role in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organisms. Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and nonribosomal peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of nonribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide synthetase genes was achieved by use of degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin microcystin was shown for both cultured and uncultured samples. Blot hybridizations, DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in cyanobacteria. The results demonstrate a molecular approach to assessing preexpression microbial functional diversity in uncultured cyanobacteria. The nonribosomal peptide biosynthetic pathways detected may lead to the discovery and engineering of novel antibiotics, immunosuppressants, or antiviral agents.

Quantitative immunofluorescence, anti-ras p21 antibody specificity, and cellular oncoprotein levels.

A general approach to investigating specificity and saturation of antibodies by quantitative immunofluorescence is applied to monoclonal antibodies generated against p21 or ras oligopeptides to quantify ras p21 oncoprotein in cultured cells. Ras 10, a panreactive mouse monoclonal antibody, appears to be a superior probe for detection of p21 in cell extracts or fixed cells because it binds a 21 kD protein on SDS-PAGE/western blots and labels the cytoplasmic membrane in a saturable and competitive manner. RAP-5, a widely used mouse monoclonal antibody generated against an oligopeptide of ras p21, does not recognize p21 in denaturing immunoblots or in immunofluorescence of cultured cells.

Is estradiol cardioprotection a nitric oxide-mediated effect?

BACKGROUND: Estradiol exerts a number of biological effects that support extensive observational data suggesting a protective role for estrogen in cardiovascular disease prevention. These include effects on lipid and carbohydrate metabolism, coagulation/fibrinolysis as well as a possible effect on vascular reactivity. It has been proposed that this might be mediated by vascular endothelial nitric oxide (NO) production. Accordingly, we designed complementary in-vivo and in-vitro studies to investigate this hypothesis further. METHODS: Firstly, in a group of 10 healthy post-menopausal women, bilateral venous occlusion plethysmography was used to examine forearm vasoconstrictor responses to intrabrachial N(G)-monomethyl-l-arginine (l-NMMA; a substrate inhibitor of nitric oxide synthase) both before and after 4 weeks of treatment with transdermal 17beta-estradiol (E(2)) (80 microg/day). Secondly, we examined the direct effects of acute (24 h) and chronic (7 days) treatment with E(2) (10 pmol/l and 10 nmol/l) on endothelial nitric oxide synthase (eNOS) gene expression in cultured human aortic endothelial cells. RESULTS: No significant differences were observed between the vasoconstrictor responses to l-NMMA (2, 4, 8 micromol/min) before and after E(2) treatment. Comparison of E(2)-treated endothelial cells with control cells showed no significant increase in eNOS mRNA expression following either acute or chronic estradiol treatment. CONCLUSIONS: The present studies do not provide evidence for an eNOS-mediated cardioprotective response to estrogen and therefore suggest that additional mechanisms other than the endothelial NO system may have an important role in the cardiovascular effects of estrogen.