Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Inhibition of carcinoma cell growth and metastasis by a vesicular stomatitis virus G-pseudotyped retrovector expressing type I insulin-like growth factor receptor antisense.

A replication-defective, vesicular stomatitis virus G-pseudotyped, Moloney murine leukemia virus retroviral vector (vLTR-IGF-IR(AS)) was generated in which a type I insulin-like growth factor receptor (IGF-IR) antisense fragment is expressed in a bicistronic mRNA with an enhanced green fluorescent protein (EGFP) reporter under the control of a potent long terminal repeat (LTR). The suitability of these retroparticles for gene therapy was tested with highly metastatic, carcinoma H-59 cells, which depend on IGF-IR expression for tumorigenicity and metastasis. Transduction with these, but not with control retroviral particles expressing EGFP only, resulted in a 70% reduction in IGF-IR levels and the loss of IGF-IR-regulated functions. Moreover, the ability of vLTR-IGF-IR(AS) retroparticle-transduced tumor cells to form experimental hepatic metastases was significantly reduced relative to controls. The results identify retrovector-mediated delivery of IGF-IR antisense as a potential strategy for cancer gene therapy.

Retrovector encoding a green fluorescent protein-herpes simplex virus thymidine kinase fusion protein serves as a versatile suicide/reporter for cell and gene therapy applications.

Expression vectors encoding herpes simplex virus thymidine kinase (HSVTK) have been extensively used in cell and gene therapy applications either as anticancer "suicide" or as "self-destruct" transgenes in adoptive immunotherapy applications. In both gene therapy applications, reliable detection of HSVTK transgene expression is required in genetically engineered cells. Direct fluorescent labeling of the HSVTK protein may be the remedy. We designed a retrovector encoding a chimeric GFP-HSVTK fusion protein that can serve as a bifunctional suicide and reporter transgene. The fusion gene was incorporated in a VSV G-pseudotyped retrovector (vGFPTKfus) and high-titer stable retroviral producer was generated ( approximately 3 x 10(6) retroparticles/ml). Tumor cell lines transduced at an MOI of 8 for 3 days led to >90% gene transfer efficiency. Southern blot analysis confirmed that unrearranged proviral genomes integrated in chromosomal DNA. Protein extract immunoblot with HSVTK antisera revealed the presence of a 70-kDa protein consistent with the predicted size of an HSVTK-GFP fusion protein. Fluorescence microscopy and FACS analysis revealed that GFPTKfus-mediated fluorescence was nuclear localized and was 30-fold greater than that observed in a bicistronic HSVTK-GFP vector. Growth of cell lines expressing vGFPTKfus was significantly suppressed in the presence of ganciclovir. The DA3 mouse mammary carcinoma cell line was transduced with vGFPTKfus and implanted in syngeneic BALB/c mice. Preestablished tumors completely regressed in seven of nine mice treated with ganciclovir. Normal human peripheral blood T lymphocytes were transduced with vGFPTKfus and nucleus-restricted green fluorescence was observed. Sorting of green fluorescent lymphocytes allowed for selection of engineered cells. In conclusion, we demonstrate the utility of vGFPTKfus as a suicide/reporter transgene in tumor cells in vitro and in vivo. Furthermore, its potential use as an analytical and therapeutic tool targeting human T lymphocytes is shown.

Glucocorticoid-inducible retrovector for regulated transgene expression in genetically engineered bone marrow stromal cells.

Transplantable bone marrow stromal cells can be utilized for cell therapy of mesenchymal disorders. They can also be genetically engineered to express synthetic transgenes and subsequently serve as a platform for systemic delivery of therapeutic proteins in vivo. Inducible production of therapeutic proteins would markedly enhance the usefulness of stromal cells for cell therapy applications. We determined whether synthetic corticosteroid hormones can be used to tightly control transgene expression via the glucocorticoid response pathway in primary bone marrow stromal cells. This regulatory mechanism does not require the presence of potentially immunogenic prokaryotic or chimeric "Trans-activators." Further, synthetic corticosteroids are pharmaceutical agents that can be readily used in vivo. We designed a self-inactivating retroviral vector in which expression of the green fluorescent protein (GFP) reporter is controlled by a minimal synthetic promoter composed of five tandem glucocorticoid response elements upstream of a TATAA box. Vesicular stomatitis virus G-pseudotyped retroparticles were synthesized and utilized to transduce cultured cell lines and primary rat bone marrow stromal cells. We have shown that primary rat bone marrow stromal cells could be efficiently engineered with our GRE-containing retrovector, basal reporter expression was low in the absence of exogenous synthetic corticosteroids, and GFP expression was dexamethasone inducible and reversible. To summarize, this strategy allows dexamethasone-induced, "on-demand" transgene expression from transplantable genetically engineered bone marrow stromal cells.

Taurine indirectly increases [Ca]i by inducing Ca2+ influx through the Na(+)-Ca2+ exchanger.

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca2+ and Na+. These results led us to believe that the increase in Na+ by taurine could be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through Na(+)-Ca2+ exchange. Therefore, we investigated the effect of beta-alanine, a blocker of the taurine-Na+ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2',4'-dimethylbenzamil), a Na(+)-Ca2+ exchanger blocker on taurine-induced [Ca]i increase in embryonic chick heart cells. Using Fura-2 Ca2+ imaging and Fluo-3Ca2+ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]i at both the cytosolic and the nuclear levels. Preexposure to 500 microM of the blocker of the taurine-Na+ cotransporter, beta-alanine, prevented the amino acid-induced increase of total [Ca]i. On the other hand, application of beta-alanine did not reverse the action of taurine on total [Ca]i. However, low concentrations of the Na(+)-Ca2+ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of beta-alanine). Thus, the effect of taurine on [Ca]i in heart cells appears to be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through the Na(+)-Ca2+ exchanger.

Endothelin-1 and insulin activate the steady-state voltage dependent R-type Ca2+ channel in aortic smooth muscle cells via a pertussis toxin and cholera toxin sensitive G-protein.

In single rabbit aortic smooth muscle cells, and at a concentration known to induce a maximum sustained increase of intracellular Ca2+ via activation of the steady-state voltage dependent R-type Ca2+ channels, endothelin-1 (10(-7) M) and insulin (80 microU/ml) were found to induce a sustained increase in cytosolic free Ca2+ ([Ca]i) levels that was significantly attenuated by pre-treatment with either pertussis toxin (PTX), cholera toxin (CTX) or removal of extracellular Ca2+. However, both PTX and CTX failed to inhibit the sustained depolarization-evoked sustained Ca2+ influx and [Ca]i elevation via activation of the R-type Ca2+ channels. Moreover, ET-1 and insulin-evoked sustained increases in Ca2+ influx were not attenuated by the selective PKC inhibitor, bisindolylmaleimide (BIS), or the specific L-type Ca2+ channel blocker, nifedipine, but were completely reversed by the R-type Ca2+ channel blocker, (-) PN 200-110 (isradipine). These data suggest that both insulin and ET-1 activate the nifedipine-insensitive but isradipine-sensitive steady state voltage dependent R-type Ca2+ channels present on rabbit VSMCs and these channels are directly coupled to PTX and CTX sensitive G protein(s).

The use of confocal microscopy in the investigation of cell structure and function in the heart, vascular endothelium and smooth muscle cells.

In recent years, fluorescence microscopy imaging has become an important tool for studying cell structure and function. This non invasive technique permits characterization, localisation and qualitative quantification of free ions, messengers, pH, voltage and a pleiad of other molecules constituting living cells. In this paper, we present results using various commercially available fluorescent probes as well as some developed in our laboratory and discuss the advantages and limitations of these probes in confocal microscopy studies of the cardiovascular system.

Bradykinin activates R-, T-, and L-type Ca2+ channels and induces a sustained increase of nuclear Ca2+ in aortic vascular smooth muscle cells.

The mechanism(s) fo Ca2+ entry stimulated by bradykinin (BK) and the receptor subtype responsible for this effect were examined in human and rabbit aortic vascular smooth muscle cells (VSMCs). Using the whole-cell voltage clamp technique, BK (10(-6)M) significantly (p < 0.05) increased both T- and L-type Ca2+ currents (ICa) in rabbit aortic VSMCs. Using the fura-2 total intracellular Ca2+ ([Ca]i) measurement technique, BK (10(-6) M) induced a transient increase of [Ca]i followed by a sustained component. Pretreatment of rabbit VSMCs with sarcoplasmic reticulum (SR) Ca2+ releaser caffeine (1-5 mM) significantly decreased the BK-induced transient increase of [Ca]i without affecting the sustained component induced by this hormone. This sustained phase was blocked by extracellular application of the Ca2+ chelator EGTA. Using the fluo-3 confocal microscopy Ca2+ measurement technique to localize cytosolic ([Ca]c) and nuclear ([Ca]n) free Ca2+ distribution, the resting sustained concentration of Ca2+ in the cytoplasm of rabbit and human aortic VSMCs was less than that in the nucleus. BK (10(-7) M) induced a nonsignificant sustained increase of [Ca]c but significant (p < 0.05) sustained increase of [Ca]n that was reversed but not prevented by the specific B1 receptor antagonist R126 (10(-6) M) as well as by the B2 receptor antagonist R817 (10(-6) M). In both VSMC preparations, the specific B1 agonist R211 (10(-9) to 10(-7) M) rapidly induced a nonsignificant increase of [Ca]c but a significant (p < 0.05) sustained increase of [Ca]n that was prevented but not reversed by the B1 selective antagonist R126 (10(-6) M). The sustained increase of [Ca]c and [Ca]n induced by BK and B1 receptor agonist was blocked by extracellular application of EGTA. These results strongly suggest that B1 and probably B2 receptors are functional in human and rabbit aortic VSMCs. BK-induced transient increase of [Ca]i is mainly due to the stimulation of T- and L-type Ica as well as to Ca2+ release from caffeine- and ryanodine-sensitive Ca2+ pools. The sustained component induced by the hormone or the B1 agonist is mainly nuclear and is due to the stimulation of Ca2+ influx through the R-type Ca2+ channels that are present at the sarcolemma and the nuclear membranes.

Modulation of cytosolic and nuclear Ca2+ and Na+ transport by taurine in heart cells.

The effect of taurine on the different types of ionic currents appears to depend on [Ca]o and [Ca]i and may also vary accordingly to tissue or cell type studied. Using microfluorometry and Ca2+ imaging techniques, short-term exposure (5-10 min) of single heart cells to taurine was found to increase total intracellular free Ca2+ in a concentration-dependent manner. However, long-term exposure of heart myocytes to taurine was found to decrease both nuclear and cytosolic Ca2+ without significantly changing either nuclear or cytosolic Na+ levels, as measured by 3-dimensional Ca2+ and Na+ confocal imaging techniques. Long-term exposure to taurine was found to prevent cytosolic and nuclear increases of Ca2+ induced by permanent depolarization of heart cells with high [K]o. This preventive effect of taurine on nuclear Ca2+ overload was associated with an increase of both cytosolic and nuclear free Na+. Thus, the effect of long-term exposure to taurine on intranuclear Ca2+ overload in heart cells seems to be mediated via stimulation of sarcolemmal and nuclear Ca2+ outflow through the Na+-Ca2+ exchanger.

Modulation of Ca2+ and Na+ transport by taurine in heart and vascular smooth muscle.

Using the whole-cell voltage clamp technique, taurine was found to affect different types of various ionic currents including T and L-type Ca2+ currents, slow Na+ and fast Na+ currents as well as the delayed outward K+ current. Also, in normal situations, taurine had no effect on the Na(+)-Ca2+ exchange current. The effect of taurine on the different types of ionic currents appears to depend on [Ca2+]o and [Ca2+]i and may also vary according to the tissue or cell type studied. Using standard Ca2+ imaging techniques, short-term exposure (10 to 20 min) of single heart cells and aortic vascular smooth muscle cells was found to increase total intracellular free Ca2+ in a dose-dependent manner. However, using 3-dimensional Ca2+ and Na+ imaging techniques, long-term exposure of heart and vascular smooth muscle cells to taurine was found to decrease both nuclear and cytosolic Ca2+ without significantly changing either nuclear or cytosolic Na+ levels. Long-term exposure to taurine was found to prevent cytosolic and nuclear increases of Ca2+ induced by permanent depolarization of heart cells with high [K+]o. This preventive effect of taurine on nuclear Ca2+ overload was associated with an increase of both cytosolic and nuclear free Na+. Thus, the effect of long-term exposure to taurine on intranuclear Ca2+ overload in heart cells seems to be mediated via stimulation of sarcolemma and nuclear Ca2+ outflow through the Na(+)-Ca2+ exchanger.

Implication of the nucleus in excitation contraction coupling of heart cells.

In the present study, Fluo-3 Ca2+ measurement and confocal microscopy techniques were used in order to localize cytosolic []c and nuclear []n free Ca2+ distribution in resting and spontaneously contracting single heart cells from 10-day-old chick embryos. In resting single cells, the concentration of Ca2+ in the cytoplasm was lower than that in the nucleus. Increasing cytosolic free Ca2+ from 100-1600 nM gradually increased [Ca2+]n with a maximum capacity near 1200 nM. Results from Fura-2 microfluorometry and Fluo-3 confocal microscopy suggest a potential cross talk between the increase of cytosolic free Ca2+ and the uptake and release of Ca2+ by the nucleus during spontaneous contraction of single myocytes. Calcium waves in spontaneously contracting cells were found to spread from one cell to the next with the nucleus acting as a fluorescent beacon in which Ca2+ levels remained elevated for several milliseconds even after cytosolic Ca2+ had returned to near basal values. These results strongly suggest that the nucleus plays a negative and positive feedback role in controlling cytosolic free Ca2+ concentration during excitation-contraction coupling in heart cells.