Faculty Bibliography

Using a ligation-mediated polymerase chain reaction technique, we have mapped the repair of ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs) at the nucleotide level in exons 1, 2, and 5 of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells. We found that CPDs are preferentially repaired in the transcribed strand (T strand) and that the order of repair efficiency is exon 1 > exon 2 > exon 5. In the cells with a deletion of the DHFR gene encompassing the promoter region and the first four exons, CPDs are not repaired in the T strand of the residual DHFR gene. These results substantiate the idea that the preferential repair of CPDs in the T strand is transcription dependent. However, in the wild type gene we have found that CPDs are efficiently repaired in the nontranscribed strand (NT strand) of exon 1 but not in the NT strand of exons 2 and 5. Probing the chromatin structure of exons 1, 2, and 5 of the DHFR gene with micrococcal nuclease revealed that the exon 1 region is much more sensitive to micrococcal nuclease digestion than the exon 2 and exon 5 regions, suggesting that the chromatin structure in the exon 1 region is much more open. These results suggest that, although preferential repair of the T strand of the DHFR gene is transcription dependent, repair of the NT strand is greatly affected by chromatin structure

BACKGROUND: We previously used a three-dimensional (3D) reconstituted basement membrane (rBM) assay to demonstrate that tumorigenic HMT-3522 T4-2 human breast cells can be induced to form morphologically normal structures ('reversion') by treatment with inhibitors of beta1 integrin, the epidermal growth factor receptor (EGFR), or mitogen-activated protein kinase (MAPK). We have now used this assay to identify reversion and/or death requirements of several more aggressive human breast cancer cell lines. METHODS: Breast tumor cell lines MCF7, Hs578T, and MDA-MB-231 were cultured in 3D rBM and treated with inhibitors of beta1 integrin, MAPK, or phosphatidylinositol 3-kinase (PI3K). MDA-MB-231 cells, which lack E-cadherin, were transfected with an E-cadherin cDNA. The extent of reversion was assessed by changes in morphology and polarity, growth in 3D rBM or soft agar, level of invasiveness, and tumor formation in nude mice. RESULTS: All three cell lines showed partial reversion (MCF7 the greatest and Hs578T the least) of tumorigenic properties treated with a single beta1 integrin, MAPK, or PI3K inhibitor. Combined inhibition of beta1 integrin and either PI3K or MAPK resulted in nearly complete phenotypic reversion (MDA-MB-231, MCF7) or in cell death (Hs578T). E-cadherin-transfected MDA-MB-231 cells showed partial reversion, but exposure of the transfectants to an inhibitor of beta1 integrin, PI3K, or MAPK led to nearly complete reversion. CONCLUSION: The 3D rBM assay can be used to identify signaling pathways that, when manipulated in concert, can lead to the restoration of morphologically normal breast structures or to death of the tumor cells, even highly metastatic cells. This approach may be useful to design therapeutic intervention strategies for aggressive breast cancers

The first orthogonal combinatorial synthesis of a high-purity triazine library was demonstrated. Novel triazine-based microtubule inhibitors were discovered by an efficient zebrafish embryo screening and in vitro microtubule polymerization assay.

OBJECTIVE: Monocyte chemoattractant protein (MCP)-1 is a proatherogenic factor that is responsible for approximately 60% of plaque macrophages in mouse models of atherosclerosis. We investigated whether lysophosphatidylcholine (LPC), enriched in oxidized low density lipoprotein, can modulate the expression of MCP-1 in arterial wall cells. METHODS AND RESULTS: LPC induced a 3-fold increase in MCP-1 mRNA in rat vascular smooth muscle cells (VSMCs) in a time- and dose-dependent manner. Nuclear runon analysis showed that this increase was attributable to increased MCP-1 gene transcription. There was a 2-fold increase in MCP-1 protein in the conditioned media of cells treated with LPC. LPC-associated increases of MCP-1 mRNA and protein were similar to those produced by platelet-derived growth factor-BB, a known inducer of MCP-1. Analyses of the MCP-1 promoter in transiently transfected VSMCs indicated an LPC-responsive element(s) between base pairs -146 and -261 (relative to transcription initiation). Further studies suggested that LPC-induced MCP-1 expression partially involves mitogen-activated protein kinase/extracellular signal-regulated kinase, a tyrosine kinase(s), and (to a lesser extent) protein kinase C but not the activation of the platelet-derived growth factor receptor. CONCLUSIONS: LPC stimulates MCP-1 expression at the transcriptional level in VSMCs, suggesting a molecular mechanism by which LPC contributes to the atherogenicity of oxidized low density lipoprotein

Mice of the PERA/Ei strain (PE mice) are highly susceptible to tumor induction by polyomavirus and transmit their susceptibility in a dominant manner in crosses with resistant C57BR/cdJ mice (BR mice). BR mice respond to polyomavirus infection with a type 1 cytokine response and develop effective cell-mediated immunity to the virus-induced tumors. By enumerating virus-specific CD8(+) T cells and measuring cytokine responses, we show that the susceptibility of PE mice is due to the absence of a type 1 cytokine response and a concomitant failure to sustain virus-specific cytotoxic T lymphocytes. (PE x BR)F(1) mice showed an initial type 1 response that became skewed toward type 2. Culture supernatants of splenocytes from infected PE mice stimulated in vitro contained high levels of interleukin-10 and no detectable gamma interferon, while those from BR mice showed the opposite pattern. Differences in the innate immune response to polyomavirus by antigen-presenting cells in PE mice and BR mice led to polarization of T-cell cytokine responses. Adherent cells from spleens of infected BR mice produced high levels of interleukin-12, while those from infected PE and F(1) mice produced predominantly interleukin-10. PE and F(1) mice infected by polyomavirus responded with increases in antigen-presenting cells expressing B7.2 costimulatory molecules, whereas BR mice responded with increased expression of B7.1. Administration of recombinant interleukin-12 along with virus resulted in partial protection of PE mice and provided complete protection against tumor development in F(1) animals.

It is important to determine how the signaling pathways for the short-term effects of nicotine (catecholamine secretion, phosphorylation of tyrosine hydroxylase) differ from those required for changes in gene expression. Our aim was to distinguish the pathways involved in short- and long-term nicotinic stimulation. PC12 cells were treated with several concentrations of nicotine from 10 micro M to 1 mM. All elicited a rapid and transient rise in [Ca(2+)](i), which was concentration dependent. After several minutes of continued exposure, a second smaller sustained rise in [Ca(2+)](i) was only observed with intermediate concentrations of nicotine (50-200 micro M). This sustained rise was not observed in cells pretreated with alpha-bungarotoxin (alpha-BTX). alpha-BTX also prevented the elevation of tyrosine hydroxylase mRNA by nicotine. The effects of brief and prolonged treatment with nicotine on the signaling pathways involved in changes in [Ca(2+)](i) and induction of tyrosine hydroxylase gene expression are summarized. The results indicate that nicotine may elicit different signaling pathways depending on the concentration. The sustained elevation of [Ca(2+)](i) via activation of alpha7 nAChRs is proposed as the mechanism leading to increased tyrosine hydroxylase gene expression.

Retinal neovascularization is a leading cause of human blindness. However, little is known concerning the molecular mechanisms controlling retinal neovascularization in vivo. Here we provide evidence that exposure of a collagen type IV cryptic epitope detected by monoclonal antibody (mAb) H

Simulated supracondylar fractures were created proximal to posterior cruciate ligament-retaining total knee arthroplasty components in paired human cadaver femora and stabilized with either a retrograde-inserted locked supracondylar nail or the Less Invasive Stabilization System (LISS; Synthes USA, Paoli, PA). Loads were applied to create bending and torsional moments on the simulated fracture stabilized with either no gap or a 10-mm gap. The LISS exhibited less torsional stability with anterior (P<.001) and posterior loads (P<.01). When varus loads were applied to 10-mm-gap specimens, the specimens stabilized with a retrograde nail had an 83% reduction in fracture displacement (P<.001) and 80% less medial translation of the distal fragment (P<.001). The samples stabilized with the LISS had a 93% reduction in fracture gap displacement when a valgus load was applied with a 10-mm gap (P<.001). Overall, these results suggest that the retrograde-inserted nail may provide greater stability for the management of periprosthetic supracondylar femur fractures in patients with a posterior cruciate ligament-retaining femoral total knee arthroplasty component