Differential expression of mammalian TRP homologues across tissues and cell lines

Mammalian homologues of the Drosophila trp gene have been invoked as the structural basis for the currents associated with capacitative Ca2+ entry (CCE) in many cell types. Trp homologues are members of a large protein family that may associate as channel subunits providing an explanation for the functional diversity of store-operated channels observed in these cells. However, there is little information as to which of these genes are co-expressed at the cellular level. We have examined the tissue specific expression of five mammalian trp genes and determined which are co-expressed in five different cell lines. The results show tissue- and cell-specific co-expression of multiple trp forms. This implies that the subunit composition of a particular CCE channel may vary depending on the cell type.     

The murine Tcl1 oncogene: embryonic and lymphoid cell expression

In human leukemias and lymphomas nonrandom chromosomal rearrangements cause changes in cell growth and/or survival in such a way as to promote malignancy. The detailed study of the biochemical and genetic pathways altered in human cancer requires the identification or development of models to allow the study and manipulation of cancer gene function. Recently, the breakpoint gene TCL1, involved in chromosome translocations observed mostly in mature T-cell proliferations and chronic lymphocytic leukemias (CLL), was isolated and characterized, and showed to be part of a new gene family of proteins involved in these tumors. The murine Tcl1 gene, is similar in sequence to the murine and human MTCP1 gene also involved in T cell leukemias. The murine Tcl1 gene was shown to reside on mouse chromosome 12 in a region syntenic to human chromosome 14. Furthermore, we show that the murine Tcl1 gene is expressed early in mouse embryonic development and demonstrates expression in fetal hematopoietic organs as well as in immature T and B cells. Characterization of the murine Tcl1 gene will help in developing a mouse model of CLL and would provide the best opportunity to study and decipher the role of TCL1 in malignant transformation.     

Mirror expression of adrenoleukodystrophy and adrenoleukodystrophy related genes in mouse tissues and human cell lines

In memory-discrimination learning, reward-produced memories are differentially rewarded such that they are the only stimuli available to support discriminative responding. Memory-discrimination learning was used in this study as follows: Reward-produced memories that were assumed to regulate instrumental performance in previously reported extinction and discrimination learning investigations were isolated and explicitly differentially reinforced (prior to a shift to extinction) in each of 4 runway investigations with rats. Results obtained here in the explicit discrimination learning stage and in the subsequent extinction stage were consistent with the prediction of the memory view and with prior discrimination learning and extinction findings. The memory interpretation was applied to memory-discrimination learning, to extinction, and to 2 other types of discrimination learning. It appears that a theory must use reward-produced memories to explain all 4 types of discrimination learning.     

Different fibroblast growth factor 1 (FGF-1) transcripts in neural tissues, glioblastomas and kidney carcinoma cell lines

The significant factors in the development of the neurosurgery program at the University of Florida have been the funding for 10 endowed chairs and a Brain Institute, the achievement of departmental status in the College of Medicine, the collaborative research with a strong Department of Neuroscience, and the strong commitment by the faculty to subspecialty neurosurgery and to service in the national neurosurgical organizations.     

Up-regulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines

Inactivation of 5-HT1A and [3H]5-HT binding sites by N-Ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ) was studied in regions of rat brain. After exposure to EEDQ (4 mg/kg body wt.) for 7 days, it is observed that the density of 5-HT1 receptor sites was decreased by nearly 20% in both cortex and hippocampus. The decrease, however, in 5-HT1A sites was more significant (70%) in both the regions. The affinity of [3H]5-HT to 5-HT1 sites was decreased significantly in both cortex and hippocampus after exposure to EEDQ, without affecting the Kd of 5-HT1A sites. Displacement studies suggested that EEDQ has high affinity to 5-HT1 sites with a Ki of 42.9+/-2.4 nM. After exposure neither basal nor 5-HT stimulated adenylyl cyclase activity was changed in cortex. The results of this study suggest that EEDQ decreases the density of 5-HT1 and 5-HT1A receptor sites but does not cause functional downregulation of these sites in rat brain.     

A nucleotide polymorphism in ERCC1 in human ovarian cancer cell lines and tumor tissues

We studied the DNA sequence of the entire coding region of ERCC1 gene, in five cell lines established from human ovarian cancer (A2780, A2780/CP70, MCAS, OVCAR-3, SK-OV-3), 29 human ovarian cancer tumor tissue specimens, one human T-lymphocyte cell line (H9), and non-malignant human ovary tissue (NHO). Samples were assayed by PCR-SSCP and DNA sequence analyses. A silent mutation at codon 118 (site for restriction endonuclease MaeII) in exon 4 of the gene was detected in MCAS, OVCAR-3 and SK-OV-3 cells, and NHO. This mutation was a C-->T transition, that codes for the same amino acid: asparagine. This transition converts a common codon usage (AAC) to an infrequent codon usage (AAT), whereas frequency of use is reduced two-fold. This base change was associated with a detectable band shift on SSCP analysis. For the 29 ovarian cancer specimens, the same base change was observed in 15 tumor samples and was associated with the same band shift in exon 4. Cells and tumor tissue specimens that did not contain the C-->T transition, did not show the band shift in exon 4. Our data suggest that this alteration at codon 118 within the ERCC1 gene, may exist in platinum-sensitive and platinum-resistant ovarian cancer tissues.     

United States population data on the multiplex short tandem repeat loci--HUMTHO1, TPOX, and CSF1PO--and the variable number tandem repeat locus D1S80

Allele frequencies for three tetrameric short tandem repeat (STR) loci HUMTHO1, TPOX, and CSF1PO and a variable number tandem repeat locus D1S80 were determined in United States Caucasian, African American, and Hispanic sample populations. All loci, except the TPOX locus in the Caucasian sample population, meet Hardy-Weinberg expectations. There is no evidence for association of alleles among the four loci. The allelic frequency data are similar to other comparable data within the same major population group.     

Differential expression of the p27Kip1 mRNA in IFN-sensitive and resistant cell lines

STUDY OBJECTIVES: In a previous study published by our group, six out of nine subjects with mild allergic asthma were shown to have an enhanced response to allergen challenge following a 1-h exposure in an 0.8-m3 exposure chamber (modified from a body plethysmograph) to an average of 120 parts per billion (ppb) ozone at rest. Other studies failed to confirm this effect. In the present study, using a similar design, we reexamined this effect using a larger group of asthmatics and a larger chamber allowing minimal fluctuations in ozone levels during exposures. DESIGN: Prospective, randomized single-blinded crossover study. SETTING: Pulmonary function laboratory equipped with an exposure chamber. SUBJECTS: Fifteen subjects had mild allergic asthma; 9 men and 6 women; the mean (SD) age was 32.5 (10) years; FEV1 was 3.4 (0.8) L; baseline methacholine provocation concentration causing a 20% fall in FEV1 was (PC20) 3.28 (4.1) mg/mL. INTERVENTIONS: Each participant was exposed, at rest, on 1 day to filtered air and on another day to ozone (mean level=120 ppb) in a larger exposure chamber than the one used in our first study with less variability in ozone level (110 to 130 vs 85 to 175 ppb) using a random, single-blinded design. After each exposure, the subject was challenged with allergen (nine with grass pollen extract and six with ragweed extract) and allergen PC15 was measured. RESULTS: Ozone preexposure did not affect allergen PC15 when compared with clean air preexposure (allergen PC15 dilution 1/114 vs 1/119, respectively). Ozone vs air preexposure resulted in an allergen PC15 that was lower in five subjects, higher in six, and unchanged (within one doubling dose) in four. CONCLUSIONS: At this low level with less variability and lower peaks than our previous study, ozone had no significant effect on airway allergen responsiveness.     

Induction of ICAM-1, HLA-DR molecules by IFN-gamma and oncogene expression in human bladder cancer cell lines

This review article describes the different aetiological agents known or suspected to cause colitis in the adult horse, namely Salmonella spp., Clostridium spp., Ehrlichia risticii, Cyathostomes, fungi, various antibiotics, drugs, and toxins, with emphasis on their mechanism of action. For each of the infectious agents, diagnostic procedures are indicated. The effects of endotoxin can be important in all forms of equine colitis.     

Absence of Fhit protein in primary lung tumors and cell lines with FHIT gene abnormalities

Genomic alterations and abnormal expression of the FHIT gene at 3p14.2 have been observed in cell lines and primary tumors of the lung. To correlate FHIT locus DNA and RNA lesions with effects on Fhit protein expression, we have analyzed 11 lung cancer cell lines, 15 small cell lung carcinomas, and 38 pairs of non-small cell primary tumors and bronchial mucosa specimens by molecular genetic and immunocytochemical methods. Using specific antibodies against the Fhit protein, we observed concordance between RNA abnormalities and lack of Fhit protein expression in lung tumors and cell lines. In addition, absence of Fhit protein in some precancerous dysplastic lesions suggested that FHIT inactivation may occur at an early phase of lung carcinogenesis.     

Cytotoxic effects of topotecan combined with various anticancer agents in human cancer cell lines

BACKGROUND: Topotecan (TPT) is a topoisomerase I poison that exhibits antineoplastic activity. Analysis of the cytotoxic effects of combinations of TPT and other anticancer agents has been limited. PURPOSE: We assessed the cytotoxic effects produced by combinations of TPT and other antineoplastic agents in experiments involving multiple human cancer cell lines of diverse histologic origins. METHODS: The cytotoxic effects of various antimetabolites (fluorouracil, methotrexate, or cytarabine), antimicrotubule agents (vincristine or paclitaxel [Taxol]), DNA alkylating agents (melphalan, bis[chloroethyl]nitrosourea [BCNU], or 4-hydroperoxycyclophosphamide [4HC]), and a DNA-platinating agent (cisplatin), alone and in combination with TPT, were measured in clonogenic (i.e., colony-forming) assays. HCT8 ileocecal adenocarcinoma, A549 non-small-cell lung carcinoma, NCI-H82ras(H) lung cancer, T98G glioblastoma, and MCF-7 breast cancer cell lines were used in these assays. The data were analyzed by the median effect method, primarily under the assumption that drug mechanisms of action were mutually nonexclusive (i.e., completely independent of one another). For each level of cytotoxicity (ranging from 5% to 95%), a drug combination index (CI) was calculated. A CI less than 1 indicated synergy (i.e., the effect of the combination was greater than that expected from the additive effects of the component agents), a CI equal to 1 indicated additivity, and a CI greater than 1 indicated antagonism (the effect of the combination was less than that expected from the additive effects of the component agents). RESULTS: When the mechanisms of drug action were assumed to be mutually nonexclusive, virtually all CIs for combinations of TPT and either antimetabolites or antimicrotubule agents revealed cytotoxic effects that were less than additive. The CIs calculated at low-to-intermediate levels of cytotoxicity for combinations of TPT and the DNA alkylating agents melphalan, BCNU, and 4HC also showed drug effects that were less than additive; in most cases, however, nearly additive or even synergistic effects were observed with these same drug combinations at high levels of cytotoxicity (i.e., at > or = 90% inhibition of colony formation). Results obtained with combinations of TPT and cisplatin varied according to the cell line examined. With A549 cells, less than additive effects were seen at low-to-intermediate levels of cytotoxicity, and more than additive effects were seen at high levels of cytotoxicity. With NCI-H82ras(H) cells, synergy was observed over most of the cytotoxicity range. CONCLUSIONS AND IMPLICATIONS: TPT cytotoxicity appears to be enhanced more by combination with certain DNA-damaging agents than by combination with antimetabolites or antimicrotubule agents. Interactions between TPT and other drugs can vary depending on the cell type examined. Further investigation is required to determine the basis of the observed effects and to determine whether these in vitro findings are predictive of results obtained in vivo.     

The expression of stem cell factor and its receptor, c-kit in human endometrium and placental tissues during pregnancy

Stem cell factor (SCF) and its receptor Kit regulate the proliferation and survival of early hematopoietic cell types as well as germ cells and melanocytes. As SCF augments the effects of several hematopoietic growth factors that are produced in reproductive tissues during pregnancy and also plays an important role in cell migration, proliferation, and survival, we studied the expression and localization of this receptor/ligand in human endometrial and placental tissues. Kit was detected by Western blot analysis in early decidual and placental tissues (8-19 weeks gestation) and in term placenta. Immunohistochemistry localized Kit mainly in trophoblast and to a lesser extent in scattered cells in the placental villous core and decidual stroma. Ribonuclease protection assay showed that SCF messenger ribonucleic acid (mRNA) expression increased 3-fold in decidua from early pregnancy compared to proliferative and secretory endometrium (P < 0.05). Placental tissues expressed 4- to 8-fold higher levels of SCF mRNA compared to decidus (P < 0.05). Isolated placental villous core expressed 7-fold higher levels of SCF mRNA than did trophoblast (P < 0.05). Thus, SCF and its receptor Kit are expressed in human endometrium and placental tissues during pregnancy, and the pattern of receptor/ligand expression suggests that endometrial and placental villous core SCF may have a paracrine effect on trophoblast through the receptor Kit.     

Heterogeneous expression of immunotherapy candidate proteins gp100, MART-1, and tyrosinase in human melanoma cell lines and in human melanocytic lesions

In recent years, it has become evident that T cells can recognize peptides of melanocytic lineage antigens such as gp100, MART-1, and tyrosinase at the tumor cell surface and can subsequently destroy these cells. It is thus feasible to develop immunotherapeutic approaches based on the melanocytic marker profiles of melanoma cells. One of the predictors of the success rate of such a treatment is the extent of positive (target) tumor cells within the lesions of the patient. First, we investigated the presence of these three proteins in 18 human melanoma cell lines using RT-PCR and immunohistochemistry. In 11 cell lines, mRNA and protein of all three markers could be detected; in one cell line, only two markers were present, and six melanoma cell lines showed no evidence for these markers. Secondly, we stained frozen sections of 105 human melanocytic lesions, 13 common nevocellular nevi, 13 atypical nevi, 13 early primary melanomas (Breslow < 1.5 mm), 25 advanced primary melanomas (aPM; Breslow > or =1.5 mm), and 41 melanoma metastases (MM) with antibodies against glycoprotein 100, melanoma antigen recognized by T cells, and tyrosinase. In addition, we used the 3,4-dihydroxy-L-phenylalanine reaction to detect tyrosinase enzyme activity as a confirmation of the tyrosinase immunohistochemical results in a subset of the lesions. In the benign lesions, glycoprotein 100 was more prominently expressed in epidermal melanocytes, whereas melanoma antigen recognized by T cells was encountered in all or nearly all dermal melanocytes in all nevocellular nevi and atypical nevus lesions. Tyrosinase was found in a lower percentage of melanocytes, both in the epidermis and in the dermis within these lesions. With regard to heterogeneity of staining within the malignant lesions, we found that 54% (early primary melanomas), 48% (aPMs), and 56% (MM) of the lesions stained within the same staining category for all three proteins studied. Approximately 17% of the aPM and MM lesions did not show positive tumor cells for any of the three proteins. We conclude that a subgroup of patients with high expression should be selected for immunotherapeutic treatment approaches based on the presence of these proteins.     

Recombinant murine cell lines, transformed by various vectors based on bovine type 1 papillomavirus and expressing human tissue plasminogen activator. II. Analysis of cell lines, producing recombinant tissue plasminogen activator

We have characterized a number of recombinant cell lines established with BPV1 and Lx1 (containing duplication of LCR-E6-E7 sequence) vectors on the basis of C127 cells. It had been shown that Lx1 based vectors possess the higher number of intracellular copies than analogous vectors on the basis of wtBPV, and most part of them is integrated into the host genome. Using various concentrations of heavy metal salts we have developed the optimized procedure for induction of recombinant tPA synthesis which is controlled by the mouse MT1 promoter. A 8-fold increase of rtPA concentration was reached in the course of induction. It had been shown that native and non-glucosylated forms of recombinant and human tPA are identical in their properties.     

HIV-1 gene expression and replication in neuronal and glial cell lines with immature phenotype: effects of nerve growth factor

Encephalopathy and neurological disorders are a major manifestation of pediatric AIDS. Although HIV-1 can replicate in cells of neuronal and glial origin, it is yet unclear whether immature neural cells, which are present during nervous system development, can support HIV-1 replication and whether neurotrophic factors can modulate HIV-1 gene expression. In this study we show that a glial cell line with a phenotype closely resembling immature glial cells is more permissive to HIV-1 infection and replication than a neuroblastic cell line. After HIV-1 infection or after transfection of these cells with the HIV-1 LTR-CAT reporter gene alone or in the presence of Tat, both HIV-1 replication and viral gene expression progressively decrease in the neuronal cell line, while they increase in the glial cell line. In both cell types viral gene expression and replication are augmented by the addition to the cells of nerve growth factor (NGF) at concentrations which induce neuronal differentiation. However, these effects are again more evident with the glial cell type, suggesting that immature glial cells may represent one of the major targets and reservoirs of HIV-1 in the developing nervous system. As NGF and Tat act synergistically in inducing HIV-1 gene expression, these data also suggest that during development the presence of high levels of neural trophic factors may activate viral replication and render the CNS more susceptible to the deleterious effects of HIV-1 infection.