Faculty Bibliography

TGFbeta is secreted as part of a latent complex that is targeted to the extracellular matrix. A variety of molecules, 'TGFbeta activators,' release TGFbeta from its latent state. The unusual temporal discontinuity of TGFbeta synthesis and action and the panoply of TGFbeta effects contribute to the interest in TGF-beta. However, the logical connections between TGFbeta synthesis, storage and action are obscure. We consider the latent TGFbeta complex as an extracellular sensor in which the TGFbeta propeptide functions as the detector, latent-TGFbeta-binding protein (LTBP) functions as the localizer, and TGF-beta functions as the effector. Such a view provides a logical continuity for various aspects of TGFbeta biology and allows us to appreciate TGFbeta biology from a new perspective

We have previously shown that fibroblast growth factor homologous factor 1B (FHF1B), a cytosolic member of the fibroblast growth factor family, associates with the sensory neuron-specific channel Na(v)1.9 but not with the other sodium channels present in adult rat dorsal root ganglia neurons. We show in this study that FHF1B binds to the C terminus of the cardiac voltage-gated sodium channel Na(v)1.5 and modulates the properties of the channel. The N-terminal 41 amino acid residues of FHF1B are essential for binding to Na(v)1.5, and the conserved acidic rich domain (amino acids 1773-1832) in the C terminus of Na(v)1.5 is sufficient for association with this factor. Binding of the growth factor to recombinant wild type human Na(v)1.5 in human embryonic kidney 293 cells produces a significant hyperpolarizing shift in the voltage dependence of channel inactivation. An aspartic acid to glycine substitution at position 1790 of the channel, which underlies one of the LQT-3 phenotypes of cardiac arrythmias, abolishes the interaction of the Na(v)1.5 channel with FHF1B. This is the first report showing that interaction with a growth factor can modulate properties of a voltage-gated sodium channel

Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids. This process is terminated when a stop codon moves into the ribosomal decoding centre (DC) and is recognized by a class-1 release factor (RF). RFs have a conserved GGQ amino-acid motif, which is crucial for peptide release and is believed to interact directly with the peptidyl-transferase centre (PTC) of the 50S ribosomal subunit. Another conserved motif of RFs (SPF in RF2) has been proposed to interact directly with stop codons in the DC of the 30S subunit. The distance between the DC and PTC is approximately 73 A. However, in the X-ray structure of RF2, SPF and GGQ are only 23 A apart, indicating that they cannot be at DC and PTC simultaneously. Here we show that RF2 is in an open conformation when bound to the ribosome, allowing GGQ to reach the PTC while still allowing SPF-stop-codon interaction. The results indicate new interpretations of accuracy in termination, and have implications for how the presence of a stop codon in the DC is signalled to PTC

BACKGROUND: One of the major constraints in elucidating the mechanisms involved in the etiology of benign prostatic hyperplasia (BPH) is the lack of suitable model systems that are readily manipulable in vitro and in vivo. To address this issue, we have used murine prostatic cell lines to establish a novel in vivo model for studying prostatic cell interactions. METHODS: Luminal, basal, and smooth muscle (SM) cell lines were inoculated alone or in combinations under the renal capsule of intact or castrated male mice, and the growth and composition of prostatic tissue in the absence or presence of doxazosin was determined. RESULTS: Both the luminal and basal cell lines reconstituted prostatic tissue if co-inoculated under the renal capsule with normal SM cells, whereas none of the lines formed significant tissue when inoculated alone. Luminal cells produced and secreted prostatic secretory products. The growth of prostatic tissue formed from co-inoculation of basal and SM cells was androgen responsive. In addition, a significant reduction in prostatic tissue was noted in animals treated with doxazosin. CONCLUSION: We have established an in vivo model that uses prostatic epithelial and SM cell lines for investigating cellular interactions between epithelial and SM cells that regulate prostatic growth and function. This model will be useful for delineating the mechanisms by which prostatic cells interact and in determining the efficacy of new approaches aimed at interfering with prostatic stromal/epithelial interactions that result in abnormal cellular proliferation

Multidrug resistance (MDR) can be mediated, in part, by overexpression of P-glycoprotein (P-gp) and is characterized by broad resistance to several structurally, chemically, and pharmacologically distinct chemotherapeutic compounds. It has been hypothesized that immunological approaches to cytolysis may be used to overcome drug resistance. RV+ is a P-gp-expressing variant of the human myeloid leukemic cell line HL60 that displays a typical MDR phenotype. MDR RV+ cells displayed relative resistance to the immunotoxin (IT) HuM195-gelonin and to free rGelonin. K562 leukemia cells retrovirally infected to overexpress P-gp are also resistant to HuM195-gelonin. In addition, a monoclonal antibody capable of inhibiting the function of P-gp was able to partially reverse resistance to the IT. These data indicated that the expression of P-gp may contribute to IT resistance in RV+. Resistance to the IT was not mediated through decreased binding to cells, nor reduced internalization into the cell because the IT displayed similar kinetics of binding and internalization for both the parental HL60 and MDR RV+ cell lines. Comparison of the cytotoxicity of other ribosome-inactivating toxins indicated that RV+ cells were not universally resistant to toxins: RV+ cells were sensitive to the actions of ricin A chain, which acts on precisely the same RNase target as gelonin. Sensitivity of the MDR RV+ cells to the protein synthesis inhibitor cycloheximide, saponin, and Pseudomonas exotoxin A additionally confirmed that the resistance was not mediated through the ribosome and that pathways downstream from the inactivation of protein synthesis leading to cell death were not substantially perturbed in the MDR cells. Resistance could be partially abrogated by bafilomycin A, which inhibits lysosomal function. Moreover, direct visualization by confocal microscopy of the intracellular trafficking route of the IT showed that the IT accumulated preferentially in the lysosome in MDR RV+ cells but not in sensitive cells. These observations implicated the process of increased lysosomal degradation as the most likely basis for resistance. Such pathways of resistance may be important in the therapeutic applications of ITs, now becoming available for human use

Carcinoma in situ (CIS) of the bladder has recently been proposed to be a heterogeneous group of diseases with varied histogenesis and biological behavior. In this study, we describe the sequential steps of CIS development and progression in a transgenic mouse model expressing low levels of the SV40 large T antigen. We found that CIS in transgenic mice arose from urothelial dysplasia, that CIS could persist for an extended period of time without invasion, and that the majority of CIS eventually evolved into high-grade, superficial, papillary tumors before a small fraction of them advanced to invasion/metastasis. A genome-wide search of chromosomal imbalances by comparative genomic hybridization revealed that 9 of 11 (82%) of CIS had losses on chromosome 11. Southern blotting demonstrated the allelic loss of the p53 gene, which resides on mouse chromosome 11, in four comparative genomic hybridization-tested tumors and 10 of 11 (91%) additional CIS examined. Consistent with the reduced p53 gene dosage because of the allelic loss and the functional inactivation of p53 protein of the remaining allele by SV40T antigen, there was a dramatic decrease in CIS of Mdm-2, a major p53 target. In contrast, the level of p21, another p53 target, was largely unaltered, suggesting that p21 expression can be regulated by p53-independent mechanisms. These results delineate the early stages of bladder tumorigenesis and suggest that the loss of a p53-bearing chromosome is an early event in bladder tumorigenesis and is crucial for the genesis and the maintenance, but not the progression, of bladder CIS. On the basis of our current and previous transgenic studies, we have proposed an integrated pathway progression model of bladder cancer