Combining serial analysis of gene expression and array technologies to identify genes differentially expressed in breast cancer.

Several methods have been used recently to determine gene expression profiles of cell populations. Here we demonstrate the strength of combining two approaches, serial analysis of gene expression (SAGE) and DNA arrays, to help elucidate pathways in breast cancer progression by finding genes consistently expressed at different levels in primary breast cancers, metastatic breast cancers, and normal mammary epithelial cells. SAGE profiles of 21PT and 21MT, two well-characterized breast tumor cell lines, were compared with SAGE profiles of normal breast epithelial cells to identify differentially expressed genes. A subset of these candidates was then placed on an array and screened with clinical breast tumor samples to find genes and expressed sequence tags that are consistently expressed at different levels in diseased and normal tissues. In addition to finding the predicted overexpression of known breast cancer markers HER-2/neu and MUC-1, the powerful coupling of SAGE and DNA arrays resulted in the identification of genes and potential pathways not implicated previously in breast cancer. Moreover, these techniques also generated information about the differences and similarities of expression profiles in primary and metastatic breast tumors. Thus, combining SAGE and custom array technology allowed for the rapid identification and validation of the clinical relevance of many genes potentially involved in breast cancer progression. These differentially expressed genes may be useful as tumor markers and prognostic indicators and may be suitable targets for various forms of therapeutic intervention.     

A single amino acid residue on the alpha(5) subunit (Ile215) is essential for ligand selectivity at alpha(5)beta(3)gamma(2) gamma-aminobutyric acid(A) receptors

Imidazobenzodiazepines such as RY-80 have been reported to exhibit both high affinity and selectivity for GABA(A) receptors containing an alpha(5) subunit. A single amino acid residue (alpha(5)Ile215) has been identified that plays a critical role in the high-affinity, subtype-selective effects of RY-80 and structurally related ligands. Thus, substitution of alpha(5)Ile215 with the cognate amino acid contained in the alpha(1) subunit (Val211) reduced the selectivity of RY-80 for alpha(5)beta(3)gamma(2) receptors from approximately 135- to approximately 8-fold compared with alpha(1)beta(3)gamma(2) receptors. This mutation produced a comparable reduction in the selectivity of RY-24 (a structural analog of RY-80) for alpha(5)beta(3)gamma(2) receptors but did not markedly alter the affinities of ligands (e.g., flunitrazepam) that are not subtype-selective. Conversely, substitution of the alpha(1) subunit with the cognate amino acid contained in the alpha(5) subunit (i.e., alpha(1)V211I) increased the affinities of alpha(5)-selective ligands by a approximately 20-fold and reduced by 3-fold the affinity of an alpha(1)-selective agonist (zolpidem). Increasing the lipophilicity (e.g., by substitution of Phe) of alpha(5)215 did not significantly affect the affinities (and selectivities) of RY-80 and RY-24 for alpha(5)-containing GABA(A) receptors. However, the effect of introducing hydrophilic and or charged residues (e.g., Lys, Asp, Thr) at this position was no greater than that produced by the alpha(5)I215V mutation. These data indicate that residue alpha(5)215 may not participate in formation of the lipophilic L(2) pocket that has been proposed to contribute to the unique pharmacological properties of alpha(5)-containing GABA(A) receptors. RY-80 and RY-24 acted as inverse agonists in both wild-type alpha(5)beta(3)gamma(2) and mutant alpha(5)I215Kbeta(3)gamma(2) receptors expressed in Xenopus laevis oocytes. However, both RY-24 and RY-80 acted as antagonists at mutant alpha(5)I215Vbeta(3)gamma(2) and alpha(5)I215Tbeta(3)gamma(2) receptors, whereas the efficacy of flunitrazepam was similar at all three receptor isoforms. The data demonstrate that amino acid residue alpha(5)215 is a determinant of both ligand affinity and efficacy at GABA(A) receptors containing an alpha(5) subunit.     

Pharmacophore/receptor models for GABA(A)/BzR subtypes (alpha1beta3gamma2, alpha5beta3gamma2, and alpha6beta3gamma2) via a comprehensive ligand-mapping approach

Pharmacophore/receptor models for three recombinant GABA(A)/BzR subtypes (alpha1beta3gamma2, alpha5beta3gamma2, and alpha6beta3gamma2) have been established via an SAR ligand-mapping approach. This study was based on the affinities of 151 BzR ligands at five distinct (alpha1-3,5,6beta3gamma2) recombinant GABA(A)/BzR receptor subtypes from at least nine different structural families. Examination of the included volumes of the alpha1-, alpha5-, and alpha6-containing subtypes indicated that region L(2) for the alpha5-containing subtype appeared to be larger in size than the analogous region of the other receptor subtypes. Region L(Di), in contrast, appeared to be larger in the alpha1 subtype than in the other two subtypes. Moreover, region L(3) in the alpha6 subtype is either very small or nonexistent in this diazepam-insensitive subtype (see Figure 16 for details) as compared to the other subtypes. Use of the pharmacophore/receptor models for these subtypes has resulted in the design of novel BzR ligands (see 27) selective for the alpha5beta3gamma2 receptor subtype. alpha5-Selective ligand 27 when injected directly into the hippocampus did enhance memory in one paradigm (Bailey et al., unpublished observations); however, systemic administration of either 9 or 27 into animals did not provide an observable enhancement. This result is in complete agreement with the observation of Liu (1996). It has been shown (Liu, 1996; Wisden et al., 1992) that in the central nervous system of the rat (as well as monkeys and pigeons) there are several native subtypes of the GABA(A) receptor which exhibit different functions, regional distributions, and neuronal locations. Although 27 binds more potently at alpha5beta3gamma2 receptor subtypes and is clearly an inverse agonist (Liu et al., 1996; Liu, 1996), it is possible that this ligand acts as an agonist at one or more subtypes. Liu (1996) clearly showed that a number of imidazobenzodiazepines were negative modulators at one subtype and agonists at another. Therefore, selectivity for a particular subtype at this point is not sufficient to rule out some physiological effect at other GABA(A)/BzR subtypes. The inability of 27 to potentiate memory when given systemically is again in support of this hypothesis, especially since alpha1beta2gamma2 subtypes are distributed throughout the brain (Wisden et al., 1992). A drug delivered systemically is far more likely to interact with all subtypes than one delivered to a specific brain region. This observation (systemic vs intrahippocampal) provides further support for the design of more subtype-specific ligands at the BzR to accurately define their pharmacology, one key to the design of new drugs with fewer side effects.     

Taxanes as radiosensitizers for head and neck cancer.

Combinations of paclitaxel and radiation therapy or paclitaxel with other chemotherapy agents and radiation have been tested with variable results in patient populations. To date, three phase I trials have been conducted using paclitaxel alone in combination with radiotherapy for the treatment of patients with head and neck cancer. Dose-limiting toxicity in the 1-hour infusion was mucositis, whereas in the 24-h/wk infusion, fever was the dose-limiting toxicity. In the long-term infusion (24 h/d, 7 d/wk), no dose-limiting toxicity was seen at the doses of paclitaxel given. In two of the protocols in which biopsies were obtained, a G2/M block was observed. A phase I protocol using paclitaxel in combination with fluorouracil and hydroxyurea with radiation and a phase II protocol using paclitaxel with cisplatin in operable head and neck cancers have been reported. Preliminary results suggest that paclitaxel in combination with radiotherapy is a reasonable experimental treatment that deserves further study in patients with stage III and IV squamous cell carcinomas of the head and neck.     

Hormone therapy: three perspectives. Fibrocystic breast disease: contraindication for oral contraceptive therapy

There exists much controversy regarding the effect of OCs (oral contraceptives) on fibrocystic breast disease. The main concern is directed to the question of whether exogenous estrogens will promote development of malignancy from the cellular atypia and hyperplasia of benign disease. Both of these conditions share many morphologic and epidemiological similarities. And, while benign breast disease occurs in younger women and breast cancer is predominantly a disease of postmenopausal Caucasian women in urban areas who have never married, it seems that the benign form does in some cases predispose a woman to the malignant form at a later date. The physiology of the breast is explained, including the mechanism of breast disease development. Mammary growth is hormonally controlled; breast disease development is estrogen-related. Tumors seem to arise at times of great estrogen supply. What is feared is that breast changes will progress from noninvasive to invasive growth, given the appropriate cellular substrate and hormonal environment. The possible latent effects of OCs should be kept in mind and considered when a physician contemplates prescribing OCs for women with fibrocystic breast disease.     

1,25-dihydroxycholecalciferol in renal osteodystrophy. Epiphysiolysis--anticonvulsant therapy

Three children with azotaemic renal osteodystrophy were treated with 1,25-dihydroxycholecalciferol (1,25(OH)2D3). All showed clinical, biochemical, and radiological improvement within 6 months of starting treatment. There were no complications. The dose of 1,25(OH)2D3 required was 0-5 microgram per day for 2 children aged 22 and 30 months, and 2 microgram per day for a 15-year-old boy. 2 of the patients were receiving phenobarbitone and phenytoin and in one of them prior treatment with dihydrotachysterol 0-5 mg daily and 6 microgram 1alpha-hydroxycholecalciferol (1alphaOHD3) daily had failed to induce improvement. In one patient, in whom serial iliac bone samples were available, 2 microgram 1,25(OH)2D3 resulted in histological improvement in previously severe osteomalacia. 1,25(OH)2D3 appears to be an effective and safe drug in the treatment of uraemic osteodystrophy.     

Assessing the feasibility of an in vitro cytotoxicity method to detect harmful ubiquitous chemicals (detection of non-warfare hazardous chemicals in the operational theater)

The objective of this work was to assess the feasibility of accomplishing aqueous extracts of soil samples and determining if the extracted solution induced adverse effects in the human myelomonocytic cell line, HL60. Dosing of HL60 cells was accomplished over a 24-hour period using 100% of extracted media from standard soil samples containing known contaminants. Assessments of viability, apoptosis, reduced thiols, and mitochondrial membrane integrity were accomplished by argon-ion laser flow cytometric analysis, using chemical labels specific for each end-point. The in vitro cytotoxicity data was compared with the results of Microtox and Mutatox tests as well as earthworm and plant toxicity tests. In vitro cytotoxicity tests' results exhibited good correlation with other tests' results.     

The characterization of human urine for specimen validity determination in workplace drug testing: a review

One challenge facing the laboratory forensic toxicologist today is verifying the validity of the random urine specimen submitted for workplace drugs of abuse analysis. Determining whether urine substitution has occurred is best accomplished through the inspection of the specimen's appearance and the performance of specific laboratory tests, such as determining the concentration of biochemical metabolic waste products and measuring indices of urine concentration. Criteria for classifying submitted urine as substituted are postulated after an extensive review of the published scientific literature. Relevant studies that were evaluated include normal random urine reference interval studies, clinical studies involving the analysis of random urine specimens, theoretical dilutional limits, medical conditions resulting in overhydration, and water-loading studies. After compilation of the study data, derived substituted criteria of urinary creatinine < or = 5.0 mg/dL and urinary specific gravity < or = 1.001 are suggested. A urine specimen meeting these criteria may be considered substituted because it is not consistent with the clinical characteristics associated with normal human urine.     

Nonstructural heart disease in the newborn. Observations during one year in a perinatal service.

One-third of 327 newborn infants referred to the perinatal service of the Hospital for Sick Children during 1975 with suspected cardiopulmonary disorders proved to have nonstructural heart disease. Most of these were term infants with transient tachypnoea or cyanosis who recovered. A history of fetal distress or difficult delivery was commonly associated. The haemodynamic disorder for most was a delay in the normal progress of the transitional circulation. Evidence of myocardial ischaemia was present in 40%, and about half of these developed congestive heart failure. Aids to diagnosis of the ischaemic complication included echocardiography and myocardial perfusion scanning. For a small proportion specific metabolic disturbances, myocarditis, or dysrhythmia seemed the primary cause but even for these there were reasonable grounds to suspect a prenatal origin. Current general supportive measures were of value in treatment.