Expression of cytochromes P450 in human breast tissue and tumors.

In an effort to determine which members of the cytochrome P450 (CYP) superfamily are expressed in human breast tissue and tumors, RNA-polymerase chain reaction studies have been undertaken. Detection of expressed CYP mRNAs identifies those forms of the enzyme that are capable of expression in breast tissue, and provides insight into the potential for in situ xenobiotic and therapeutic drug metabolism. CYP1A1 mRNA was present in (5/11) breast tissues and (6/13) tumors. When normal and tumor tissues were from the same individuals, higher amplification occurred in normal tissues. CYP1B1 mRNA was present in all but one tissue, and CYP2C mRNA forms were present in all of the tissues. CYP3A4 mRNA was present in (8/11) normal breast tissues and (2/13) tumor tissues, and CYP3A5 mRNA was present in (9/11) normal tissues and (2/13) tumor tissues. The expression of the CYP3A mRNA forms was not coincident, suggesting differential regulation. CYP2D6 mRNA was present in (10/11) normal breast tissue and (10/13) tumors. Two splice variants of CYP2D6 mRNA were also detected; one with a 207 bp intron spliced in was detected in all of the normal tissue samples and (11/13) tumors, whereas another (which lacks a 3'-portion of exon 6) was detected in (9/11) normal breast tissues and (7/13) tumors. Thus, examples of each of the xenobiotic-metabolizing CYP1, CYP2, and CYP3 subfamilies were detected in low levels in human normal breast tissue and tumors. The machinery for possible in situ bioactivation of xenobiotics and modification of therapeutic drugs is thus present in human breast tissue.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate and 17 beta-estradiol on estrogen receptor regulation in MCF-7 human breast cancer cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF-7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER-ICA), and on ER function by competitive binding assays under conditions of saturating 17 beta-estradiol (E2). Comparative studies were conducted with E2 and 12-O-tetradecanoylphorbol-13-acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E2 and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER-ICA after 72 h exposure to 1 nM E2 and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [3H]E2 under saturating conditions (10 nM E2) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [3H]E2 was depressed by 49% in MCF-7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1-h treatment with 5 microM alpha-naphthoflavone, which inhibits TCDD-induced, P450-mediated, E2 metabolism, and subsequent E2 depletion. In conclusion, while TPA and E2 effectively down-regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF-7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF-7 cells by TCDD.

Increased bcl-2 protein levels in prostatic adenocarcinomas are not associated with rearrangements in the 2.8 kb major breakpoint region or with p53 protein accumulation.

bcl-2, an inhibitor of programmed cell death (apoptosis), is present in high levels in a subset of prostatic adenocarcinomas. In this study, 42 prostatic adenocarcinomas were analyzed to determine whether increased bcl-2 levels are associated with rearrangements in the 2.8-kb major breakpoint region, an association known to occur in certain follicular lymphomas featuring a t(14:18) translocation. Immunostaining for bcl-2 and p53 proteins was performed on formalin-fixed, paraffin-embedded tumor specimens using murine anti-human bcl-2 and p53 monoclonal antibodies in all 42 cases. Genomic DNA from paired frozen samples of each tumor was subjected to digestion with HindIII and EcoRI and the products analyzed on a Southern blot with a 2.8-kb-digoxigenin-labeled major breakpoint region probe. Comparisons between groups were evaluated with the Fisher exact test. Diffuse, strong cytoplasmic immunoreactivity for bcl-2 was present in the epithelial cells of tumor glands in 16 of 42 cases (38%), including 8 of 19 low grade (Gleason score 6 and below) and 8 of 23 high grade (Gleason score 7 and above) prostatic adenocarcinoma. Southern blotting demonstrated a normal 2.8-kb germline DNA fragment in every case, with no evidence of rearrangement. Nuclear p53 staining was present in 10 of 24 high grade and 0 of 18 low grade tumors (P < 0.001). Only four cases exhibited positivity for both bcl-2 and p53, and there was no association of bcl-2 positivity with co-expression of the p53 protein (P = 0.58). We conclude that aberrations in the function of either bcl-2 or p53 could possibly modify the apoptotic pathway resulting in the extended survival of tumor cells. Also, increased bcl-2 levels in prostatic adenocarcinomas occur in the absence of detectable rearrangements in the major breakpoint region.

E-cadherin expression in papillary transitional cell carcinoma of the urinary bladder.

E-cadherin (E-CD), a cell adhesion molecule that plays a diverse role in cell-cell and cell-matrix interaction, has recently been associated with tumor invasion and metastasis. We evaluated the E-CD expression in 55 cases of urinary bladder papillary transitional cell carcinoma using a double-linked immunoalkaline phosphatase procedure on formalin-fixed, paraffin-embedded specimens quantified as percentage positive staining area with the Roche RPW image analyzer (Roche Image Analysis Systems, Elon College, NC). The 10 grade I tumors stained intensely for E-CD (66%) and showed enhanced staining compared with normal transitional epithelium. The 20 grade II lesions stained moderately at 45%, and the 25 grade III lesions showed weak expression of E-CD with a 33% positive stain. This loss of expression for high-grade versus low-grade tumors was statistically significant (P < .02). There was a similar decrease in E-CD expression for high-stage versus low-stage specimens with 30 stage 0 and stage A cases having an average of 51% positive stain and 25 stage B, C, and D cases having an average stain of 33%. This difference was also statistically significant (P < .001). We conclude that loss of expression of E-CD in high-grade, high-stage urinary bladder transitional cell carcinoma is associated with significant bladder wall invasion, indicates potential for metastasis, and may be of clinical value in the assessment of prognosis and planning of therapy for patients with bladder tumors.

Quantitative immunohistochemical determination of cathepsin D levels in prostatic carcinoma biopsies. Correlation with tumor grade, stage, PSA level, and DNA ploidy status.

National screening programs resulting in an increased detection rate of prostatic adenocarcinoma have prompted the search for new methods of predicting disease outcome that can be applied to the initial narrow bore needle biopsy specimens. Cathepsin D, a lysosomal aspartyl protease and autocrine mitogen, has been studied in a wide variety of human neoplasms as an invasion and metastasis marker. Prostatic carcinoma needle biopsy tumor cell cathepsin D content was measured in 61 men using a semiquantitative image analysis assisted immunohistochemical procedure. Results were compared with preoperative serum prostatic specific antigen levels, tumor grade, DNA ploidy status, pathologic stage after radical prostatectomy and disease recurrence during a median 2.6 year follow-up. Biopsy cathepsin D levels significantly correlated with tumor grade (P = .022) and DNA ploidy status (P = .028) by logistic regression analysis. Post-prostatectomy pathologic stage and disease recurrence did not correlate with tumor cathepsin D levels. Final prostatectomy grade and DNA ploidy status independently predicted metastasis and post-operative disease recurrence (P < .001). Although this study did not find independent prognostic status for cathepsin D in prostate cancer, the correlation with tumor grade and DNA ploidy status is noteworthy and the inter-relationship of outcome variables may prove of interest and warrant further evaluation of this potential predictor or CO-predictor of disease outcome.

Quantitative RNA-polymerase chain reaction-DNA analysis by capillary electrophoresis and laser-induced fluorescence.

Quantitative RNA-polymerase chain reaction (RNA-PCR) is an extremely powerful analytical tool owing to its specificity and high level of sensitivity. Quantitative RNA-PCR is, however, highly labor intensive. No analytical method currently exists that can accurately and rapidly quantitate the small quantities of DNA in RNA-PCR reaction mixtures. We have developed a method using capillary electrophoresis and laser-induced fluorescence to detect YOYO-1 complexes of DNA produced by PCR. RNA-PCR mixtures can be analyzed either directly (without primer and protein removal) or by electrokinetic injection following desalting. Modified competitive and multiplex competitive RNA-PCR assays for glyceraldehyde-3-phosphate dehydrogenase and P4501A1 were tested in a series of mixtures containing equal concentrations, but different proportions, of RNA from untreated (essentially no P4501A1 mRNA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated (high levels of P4501A1 mRNA) HepG2 cells. Twofold differences in concentrations between two P4501A1 mRNA solutions could be detected by competitive RNA-PCR. Glyceraldehyde-3-phosphate dehydrogenase concentrations were constant throughout. Multiplex competitive PCR produced more variable results due to the presence of contaminating peaks, which hindered accurate area integration. These data demonstrate the potential usefulness of capillary electrophoresis in a variety of quantitative PCR applications.

Alteration of the p53 locus in benign hyperplastic prostatic epithelium associated with high-grade prostatic adenocarcinoma.

Recent evidence suggests that the tumor suppressor protein, p53, protects somatic cells against the accumulation of genomic mutations. The genomes of cells lacking normal p53 function may become hypermutable, a condition that might result in the accumulation of multiple genetic alterations as the affected cells proliferate. Such cells may then become more susceptible to malignant transformation. We hypothesized that some high-grade prostate cancers might arise from foci of morphologically benign cells that had previously sustained p53 lesions. As an initial test of this hypothesis, we employed a microdissection technique to isolate morphologically benign cells within hyperplastic glands located near foci of high-grade adenocarcinoma. Genomic DNA from these cells was subjected to polymerase chain reaction amplification and single-stranded conformational polymorphism analysis for detecting alterations in the p53 locus. With use of this approach, gross alterations in the p53 locus were demonstrated in benign cells in 1 of 20 (5%) specimens harboring high-grade malignancy (Gleason grade 7 or higher). Thus, in some cases, hyperplastic prostatic epithelium harbors preneoplastic genetic alterations that could possibly give rise to high-grade malignancies.

E-cadherin expression in prostatic carcinoma biopsies: correlation with tumor grade, DNA content, pathologic stage, and clinical outcome.

We compared tumor grade and DNA content with expression of E-cadherin (E-CD), a cell adhesion molecule associated with cell-cell and cell-matrix interaction, leukocyte function, and tumor invasion and metastases, on 56 prostate carcinoma needle biopsies. The findings were correlated with final pathologic stage at subsequent prostatectomy, preoperative serum prostate-specific antigen level and further development of metastases during an initial 2.4-yr mean clinical follow-up period (range 0.5 to 5.5 yr). E-CD expression (uvomorulin, L-CAM, cell CAM 80/120, ARC-1, Sigma, St. Louis, MO) was measured by double-linked immunoalkaline phosphatase immunohistochemistry quantified with a the Roche RPW image analyzer (Roche Image Analysis Systems, Elon College, NC). DNA ploidy was determined on formalin-fixed, paraffin-embedded Feulgen-stained 5-microns tissue sections of the narrow-bore initial prostate carcinoma biopsies with the Roche RPW image analyzer. The 51% mean positive area E-CD expression in the group of 56 adenocarcinomas was significantly less than the 76% expression level for 15 normal control prostate tissues (P < 0.001). E-CD expression was also decreased in aneuploid (39%) versus diploid tumors (54%, P < 0.001); and in high-grade (44%) versus low-grade lesions (54%; P < 0.01). The 44% E-CD expression level in patients with metastases was lower than the 52% level in the nonmetastatic cases, but this finding was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Observations on tumor and metastatic suppressor gene status in endometrial carcinoma with particular emphasis on p53.

BACKGROUND: Genetic changes in the development of endometrial carcinoma have not been characterized, and little is known of tumor or metastatic suppressor gene status in these malignancies. The current study on endometrioid carcinoma was undertaken to examine the status of two tumor suppressor genes that frequently have been found to be altered in human malignancies (the p53 gene and the retinoblastoma [Rb] gene) and to examine the status of the candidate metastatic suppressor gene, nm23-H1. METHODS: The status of the p53 gene was studied by immunohistochemistry of formalin-fixed paraffin-embedded biopsy samples from 72 patients with atypical endometrial hyperplasia or endometrioid carcinoma who underwent hysterectomy immediately after biopsy and from 5 patients with benign endometria. A search for loss of heterozygosity (LOH) of the nm23 gene after DNA extraction from frozen tissues and hybridization with nm23-H1 cDNA specific probe was made in 10 endometrial carcinomas. Rb gene status was evaluated by image analysis quantification of immunoreactive retinoblastoma protein in frozen sections of 10 carcinomas and 2 benign endometria. RESULTS: p53 expression was low in all benign endometria, but high expression was found in 2 (15%) of 13 atypical hyperplasias and in 23 (39%) of 59 carcinomas. High levels of p53 expression in endometrioid carcinoma correlated with the spread of disease outside of the uterus and by logistic regression, the presence of squamous differentiation, nuclear grade, and high p53 expression in the biopsy all independently correlated with spread of disease outside of the uterus. Although 7 of the 10 carcinomas studied were informative, LOH for the nm23 gene was not seen in any, including a site of metastasis. Rb protein expression in endometrial carcinoma was similar to expression in benign endometria. CONCLUSIONS: Although this study found no evidence of nm23-H1 gene alteration or alterations in Rb protein levels in endometrial carcinoma, high expression of p53 protein was sporadically identified in biopsy specimens of atypical hyperplasia and frequently found in endometrioid carcinomas. Determination of p53 expression in combination with the presence or absence of squamous differentiation and nuclear grade in biopsy material may help predict spread of endometrioid carcinoma outside the uterus and facilitate therapeutic planning before hysterectomy.

Association of p53 immunoreactivity with high gleason tumor grade in prostatic adenocarcinoma.

To determine whether p53 immunoreactivity correlates with the Gleason tumor grade in primary adenocarcinoma of the prostate we analyzed 107 consecutive surgical specimens (78 radical prostatectomies and 29 transurethral resections). A hematoxylin-eosin-stained slide from a representative block of each tumor was examined, and primary and secondary Gleason scores were assigned in each case. Additional paraffin sections from the same block were stained immunohistochemically for p53 expression using the monoclonal antibody clone DO-1, a mouse IgG2a directed against a denaturation-resistant epitope of p53. Four of 54 (7.4%) low-grade tumors (combined Gleason score of 6 and below) and 11 of 53 (20.8%) high-grade tumors (combined Gleason score of 7 and above) revealed strong nuclear positivity for p53. When evaluated using only the primary Gleason score, none of 23 (0%) Gleason grade 2 tumors and 15 of 84 (17.9%) Gleason grade 3 or higher tumors were positive. These data demonstrate a positive association between p53 immunoreactivity and higher Gleason grade tumors (P = .04 for the combined score, P = .02 for primary score only). In addition, we noted occasional p53-positive nuclei in basal cells of benign glandular acini in regions flanking tumor. Focally positive nuclear staining also was demonstrated in basal cells from nine of 25 prostate glands exhibiting benign prostatic hyperplasia with no tumor. These results suggest that p53 overexpression might be associated with the known proliferative capacity of basal cells in benign hyperplastic prostate glands, and that mutations of p53 might play a role in the pathogenesis of a subset of high-grade prostate adenocarcinomas.

Recent advances in diabetes care and management.

Four developments in diabetes care and management have evolved over the last decade and are now routinely incorporated into clinical practice: (1) the introduction of recombinant human insulin; (2) the use of second generation oral agents; (3) home glucose monitoring and glycohemoglobin monitoring; and (4) a better understanding of the relationship between diabetic control and complications.

Treatment of snakebite poisoning.

The epidemiology, mechanics, prevention, pharmacology, clinical manifestations, and treatment of snakebites are reviewed. Poisonous snakes bite approximately 8000 persons annually in the United States, causing approximately 12-15 deaths per year. Pit vipers (rattlesnakes, copperheads, cottonmouths, and massasaugas) are responsible for 99% of all snakebite poisonings; coral snakes and other foreign exotic species are responsible for the additional 1%. Envenomation is characterized by pain, edema, and ecchymoses at or near the site of venom injection, followed by cardiac, hematologic, neurologic, renal, and pulmonary toxicity. The major clinical finding in most snakebite poisonings is local tissue necrosis. Immediate treatment for snakebite includes limiting movement and placing a constriction band proximal to the site of venom injection. If medical care is more than 30 minutes away, the wound may be incised and suctioned. Antivenin therapy is the mainstay of medical treatment of snakebite, along with administration of plasma expanders, pain medication, diazepam, tetanus toxoid, antiseptics, and antibiotics. Patients who have pain, swelling, ecchymoses, systemic symptoms, or abnormal laboratory findings within 30 minutes to one hour of a bite are probable candidates to receive antivenin therapy. Before receiving antivenin therapy, the patient must be tested for hypersensitivity to the antivenin. Antivenin therapy is most effective when given within four hours of the snakebite. Pharmacists--especially those serving rural areas--should be familiar with current snakebite treatments, both local and systemic, and should be prepared to provide important information and dispel any myths about snakebite poisoning.

Drug usage evaluation: H2-receptor antagonist use in 30 hospitals.

In this 30-hospital survey, data on usage, adverse drug reaction (ADR) rates, and drug interactions (DI) of the H2-receptor antagonists (H2RA) cimetidine, ranitidine, and famotidine were analyzed. Approximately 8% (1,768 patients) admitted to participating hospitals received an H2RA (range: 0.5 to 30%). Patients admitted to a critical care area were significantly (p less than 0.025) more likely to receive ranitidine, were more likely (p less than 0.005) to receive more than one H2RA, and were more likely (p less than 0.0005) to receive an H2RA for "stress ulcer" or a "history of peptic ulcer disease". Doses of the H2RAs were appropriate in less than 50% of cases. Sixty ADRs and 29 DIs were reported. There was no significant difference (p greater than 0.70) between cimetidine and ranitidine with regard to the number of ADRs reported; however, cimetidine was associated with a significantly greater (p less than 0.0005) number of reported DIs. The results of this utilization review indicate that H2RAs are frequently prescribed drugs in hospitalized patients. All ADRs and DIs were reported in patients in critical care areas, suggesting that these patients are "at risk" of developing side effects to H2RAs.

The effects of amiodarone on thyroid hormone function: a review of the physiology and clinical manifestations.

Amiodarone, an iodinated benzofuran derivative, is used for treatment of refractory cardiac arrhythmias. Certain features of the drug's structure resemble those of the biologically active thyroid hormone, triiodothyronine (T3). In addition, the drug has a variety of complex effects on thyroid hormone physiology, including a number of possible antagonistic effects on thyroid hormone function at the cellular level. The drug occasionally causes clinically overt hyperthyroidism and hypothyroidism. We review these effects and discuss their clinical implications.

Comparison of excretion of nicotinuric acid after ingestion of two controlled release nicotinic acid preparations in man.

We tested an inexpensive controlled-release nicotinic acid product (Bronson Pharmaceuticals, LaCanada, CA) and compared it with the standard, more expensive, controlled release product, Nicobid (Rorer Pharmaceuticals), by measuring the 24 hour urinary recovery of nicotinic and nicotinuric acids from ten subjects following 500 mg oral ingestion of each product. Nicotinuric acid is the major detoxification product of nicotinic acid and may serve as a simple quantitative index of hepatic biotransformation of nicotinic acid. Although both products demonstrated controlled release profiles, the rate of appearance of nicotinic and nicotinuric acid in the urine as well as the rate of in vitro drug dissolution of the Bronson product were more rapid compared with Nicobid. Moreover, the total amounts of nicotinic acid and nicotinuric acid recovered in the urine after 24 hours were greater for the Bronson product (P less than .05). Since sustained presentation of nicotinic acid to the liver may correlate with clinical antihyperlipidemic effects, our results suggest that the Bronson product may prove to be a clinically useful preparation.

Nicotinic acid: a review of its clinical use in the treatment of lipid disorders.

Nicotinic acid (niacin) is a water-soluble vitamin widely used for the treatment of lipid disorders. In pharmacologic doses (1 g or more/day), alone or in combination with other lipid-lowering drugs, nicotinic acid lowers very low-density (VLDL) and low-density lipoprotein (LDL) levels, while concurrently increasing high-density lipoprotein (HDL) levels. It may reduce long-term mortality in patients with known coronary artery disease and may slow or reverse the progression of atherosclerosis. A major consideration against using nicotinic acid is the occurrence of frequent, bothersome, adverse reactions such as cutaneous flushing, skin rash, and gastric upset. Careful dosing titration may, however, minimize these effects. The beneficial effects, taken together with the low cost of nicotinic acid therapy and the relative freedom from serious side effects, have made nicotinic acid the agent of choice for the treatment of many patients with hyperlipidemia.