Faculty Bibliography

'Bystander killing' is a term used to describe the broad cell death associated with the transduction of the herpes simplex virus thymidine kinase gene (HSV1-tk) and administration of nucleoside analogs and which extends the killing effect to adjacent cells not transduced with HSV1-tk ('bystander cells'). HSV1-tk negative cells can be killed by co-culture with HSV1-tk positive cells at a ratio as small as one HSV1-tk positive to 32 HSV1-tk negative cells (1:32). In this report, several aspects of bystander killing are characterized. First, the sensitivity to bystander killing is shown to differ among cell lines. Second, cell-to-cell contact, or at least proximity between cells, is demonstrated to be necessary for bystander killing. Third, forskolin is shown to inhibit bystander killing. We also show that bystander killing is not species specific. Finally, it is demonstrated that cell death induced by bystander killing is mediated via apoptosis

pH dependent absorption and fluorescence spectra of hypericin 1 and its octahydroxy analogue 3 demonstrate their acidic properties, their deprotonation occurring in the ground and excited states from OH in the bay region and in the peri position to the carbonyl, respectively; the presence of the anion of 1 in the crystalline state is established by X-ray diffraction

Hypericin is a polycyclic, aromatic, naphthodianthrone which has been shown to possess in vivo and in vitro antiretroviral activity. To gain further insight into the mechanism(s) by which hypericin exerts its antiretroviral effects, we have studied Radiation Leukemia virus (RadLV) produced from cells pulse-treated with hypericin. Hypericin-treatment did not inhibit retroviral production or the proteolytic cleavage of the gag-encoded precursor proteins. Rather, hypericin was found to be associated with RadLV particles, the retrovirions showed an increased density in sucrose, and the RadLV protein banding patterns were altered. RadLV produced from hypericin-treated cells was rendered noninfectious upon exposure to visible light. Our results suggest that RadLV produced from hypericin-treated cells is inactivated by a hypericin-mediated photodynamic process

The MHC class II I-A(s) positive B cell lymphomas reticulum cell sarcoma (RCS) that arise in > 90% of SJL mice by the age of 12 months have superantigen-like stimulating properties. In the present study, therefore, RCS cell lines were examined for abnormal expression of endogenous mouse mammary tumor virus (MMTV) proviruses. Extraordinarily high expression of a 1.8 kb mRNA hybridizing with the long terminal repeat (LTR) of MMTV was found in both primary lymphomas and in vitro RCS lines, but not in an SJL B cell lymphoma, NJ101, that does not stimulate syngeneic T cells, or in LPS activated SJL B cells. A cDNA was cloned from cRCS-2 and sequenced. A 31mer oligonucleotide probe, prepared based on the unique C-terminal sequence of this RCS-Mtv LTR, detected the 1.8 kb mRNA in all RCS lymphomas, while a similar probe for the C-terminal sequence of Mtv-8 LTR hybridized with the larger mRNA present in normal B cells and in NJ101. Preincubation with 19mer antisense S-oligonucleotides, prepared based on the sequences of the first two potential translation initiation sites common to both Mtv-8 and the RCS-Mtv LTR, significantly reduced the ability of RCS cells to stimulate syngeneic T cells. Moreover, transfection of NJ101 cells with the cloned RCS-MMTV cDNA conferred V beta 16 T cell stimulating properties on to these cells. It is concluded that expression of the product of this MMTV-LTR mRNA provides RCS with the strong T cell stimulating properties that it needs for its growth. These results thus identify a novel oncogenic property of MMTV-LTR

The murine cationic amino acid transporter is also the receptor for murine ecotropic leukemia retrovirus (MuLV-E). Recently, we have cloned a human gene (H13) homologous to the murine ecotropic retroviral receptor (ERR). Although the human homolog is very similar to murine ERR in sequence (87.6% amino acid identity) and structure (14 transmembrane-spanning domains), the human protein fails to function as a receptor for MuLV-E. To identify amino acid residues critical for MuLV-E infection, we took advantage of this species difference and substituted human H13 and murine ERR amino acid residues. Mouse-human chimeric receptor molecules were generated by taking advantage of using common restriction sites. These studies demonstrated that extracellular domains 3 and/or 4 contain the critical amino acid residues. Oligonucleotide-directed mutagenesis was then used to create 13 individual ERR mutants containing one or two amino acids substitutions or insertions within these two extracellular domains. Substitution of as few as one amino acid residue (Tyr) at position 235 in ERR with the corresponding H13 amino acid residue Pro abrogates the ability to function as a receptor for MuLV-E infection. Conversely, substitution of just two amino acid residues at positions 240 and 242 or 242 and 244 in H13 with the corresponding amino acid residues in ERR endows H13 with the ability to function as the receptor. This observation can be utilized to significantly improve the safety of retrovirus-mediated gene therapy in humans

Following attachment and entry of human immunodeficiency virus (HIV) into a host cell, the HIV genomic RNA is reverse transcribed to cDNA. This step may be inhibited by hypericin, a compound that induces alterations of the retroviral capsid. Incubation of HIV with hypericin rendered the virus noninfectious. The replication of HIV was blocked early; HIV cDNA could not be detected in cells challenged with hypericin-treated HIV. Hypericin did not inhibit the binding of recombinant gp120 to CD4+ cells, nor did hypericin inhibit syncytium formation. However, reverse transcriptase activity could not be released from hypericin-treated virions. Western blot analysis revealed altered mobility of the HIV major capsid protein (p24) following hypericin treatment. Hypericin-treated recombinant HIV p24 exhibited similar altered mobility. The inactivation of HIV infectivity and the alterations in p24 mobility required hypericin incubations in the presence of visible light. Collectively, these data suggest that photochemical alterations of the HIV capsid may contribute to the hypericin-mediated inactivation of HIV. Such alterations may inhibit the release of RT activity from treated HIV, and prevent uncoating and subsequent reverse transcription of the HIV genome within a target cell