Biomedical subjects

J Galipeau

Publications and source records attributed to J Galipeau.

At least 19 recordsLinked to original sources

PubMed2003

A neovascularized organoid derived from retrovirally engineered bone marrow stroma leads to prolonged in vivo systemic delivery of erythropoietin in nonmyeloablated, immunocompetent mice.

Marrow stromal cells (MSCs) are postnatal progenitor cells that can be easily cultured ex vivo to large amounts. This feature is attractive for cell therapy applications where genetically engineered MSCs could serve as an autologous cellular vehicle for the delivery of therapeutic proteins. The usefulness of MSCs in transgenic cell therapy will rely upon their potential to engraft in nonmyeloablated, immunocompetent recipients. Further, the ability to deliver MSCs subcutaneously - as opposed to intravenous or intraperitoneal infusions - would enhance safety by providing an easily accessible, and retrievable, artificial subcutaneous implant in a clinical setting. To test this hypothesis, MSCs were retrovirally engineered to secrete mouse erythropoietin (Epo) and their effect was ascertained in nonmyeloablated syngeneic mice. Epo-secreting MSCs when administered as 'free' cells by subcutaneous or intraperitoneal injection, at the same cell dose, led to a significant - yet temporary - hematocrit increase to over 70% for 55+/-13 days. In contrast, in mice implanted subcutaneously with Matrigel trade mark -embedded MSCs, the hematocrit persisted at levels >80% for over 110 days in four of six mice (P<0.05 logrank). Moreover, Epo-secreting MSCs mixed in Matrigel elicited and directly participated in blood vessel formation de novo reflecting their mesenchymal plasticity. MSCs embedded in human-compatible bovine collagen matrix also led to a hematocrit >70% for 75+/-8.9 days. In conclusion, matrix-embedded MSCs will spontaneously form a neovascularized organoid that supports the release of a soluble plasma protein directly into the bloodstream for a sustained pharmacological effect in nonmyeloablated recipients.

Animals↗

PubMed2003

Postnatal bone marrow stromal cells elicit a potent VEGF-dependent neoangiogenic response in vivo.

Bone marrow stromal cells (MSCs) are pluripotent cells capable of differentiation into several tissue types. This present study was performed to determine their functional neoangiogenic potential in vivo. Whole bone marrow was harvested from C57Bl/6 mice, and the adherent cellular fraction was culture expanded for 14 doublings. These MSCs were resuspended in Matrigel and their angiogenic effect assessed in isogenic recipients. At 2 weeks postimplantation, the mean vascular density in Matrigel plugs containing 2 x 10(6) MSCs/ml was 41+/-5.0 blood vessels (BVs)/mm(2) compared to 0.5+/-0.7 for empty Matrigel (P<0.001). In comparison, Matrigel plugs containing either recombinant murine VEGF 165 at 50 ng/ml or bovine bFGF at 1000 ng/ml generated 21+/-5 and 11+/-2.0 BV/mm(2), respectively. Arteriogenesis was observed only in the MSC-containing implants. With the use of LacZ retroviral labeling of ex vivo expanded MSCs, we show that approximately 10% of LacZ(+)MSCs differentiated into CD31(+) and VEGF(+) endothelial cells. More than 99% of the neoangiogenic phenomena arose from recruitment of host-derived LacZ(null) vascular structures. Neutralizing anti-VEGF antibodies inhibited the MSC-initiated angiogenic response in vivo by 85% (P<0.001). In conclusion, MSCs have the ability to effectively recruit and participate in angiogenesis and arteriogenesis de novo and VEGF plays a central role in the observed host-derived angiogenic response. We propose that ex vivo expanded autologous MSCs may serve as cell therapy to promote therapeutic angiogenesis.

Animals↗

PubMed2002

Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells.

Naturally occurring drug resistance genes of human origin can be exploited for selection of genetically engineered cells co-expressing a desired therapeutic transgene. Their non-immunogenicity in clinical applications would be a major asset. Human cytidine deaminase (hCD) is a chemoresistance gene that inactivates cytotoxic cytosine nucleoside analogs, such as cytosine arabinoside (Ara-C). The aim of this study was to establish if the hCD gene can serve as an ex vivo dominant selectable marker in engineered bone marrow stromal cells (MSCs). A bicistronic retrovector comprising the hCD cDNA and the green fluorescent protein (GFP) reporter gene was generated and used for transduction of A549 cells and primary murine MSCs. Analysis of transduced cells demonstrated stable integration of proviral DNA, more than 1000-fold increase in CD enzyme activity, and drug resistance to cytosine nucleoside analogs. In a mixture of transduced and untransduced MSCs, the percentage of retrovector-expressing cells could be increased to virtual purity (>99.5%) through in vitro drug selection with 1 microM Ara-C. Increased selective pressure with 2.5 microM Ara-C allowed for enrichment of a mixed population of MSCs expressing approximately six-fold higher levels of GFP and of CD activity when compared with unmanipulated engineered MSCs. Moreover, engraftment and endothelial differentiation of these in vitro selected and enriched gene-modified marrow stromal cells was demonstrated by Matrigel assay in vivo. In conclusion, these findings outline the potential of human CD as an ex vivo selection and enrichment marker of genetically engineered MSCs for transgenic cell therapy applications.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

A novel parasite-derived suicide gene for cancer gene therapy with specificity for lung cancer cells.

The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) expressed by the parasite Trypanosoma brucei (Tb) can convert allopurinol, a purine analogue, to corresponding nucleotides with greater efficiency than its human homologue. We have developed a retroviral system that expresses the parasitic enzyme and tested its capacity to activate the prodrug allopurinol to a cytotoxic metabolite. Cytotoxicity assays demonstrated that five non-small cell lung carcinoma cell lines transduced with the construct were sensitized to the prodrug by 2.1- to 7.6-fold compared with control values. This selectivity was not observed in seven other cell lines also expressing the construct, such as breast carcinoma. Assays indicated that enhanced cytotoxicity to allopurinol correlated with induction of apoptosis in lung cancer cells. The selectivity of this suicide gene was not explained either by the TbHGPRT expression or by the allopurinol accumulation. Our study shows that this novel system may represent a therapeutic tool for gene prodrug targeting of lung cancer, considering the fact that allopurinol is well tolerated in humans.

Allopurinol↗

PubMed2001

Parathyroid hormone-related protein promotes quiescence and survival of serum-deprived chondrocytes by inhibiting rRNA synthesis.

Parathyroid hormone-related protein (PTHrP) was initially recognized for its ability to promote parathyroid hormone-like bioactivity in kidney, bone, and squamous epithelial cells. PTHrP is a multifunctional protein in which bioactivity is mediated by two distinct pathways. Its classic parathyroid hormone-like activity results from binding of its amino terminus to cell surface PTH1R and activation of signal transduction pathways. Another less well recognized pathway involves translocation of PTHrP to the nucleus via a mid-region bipartite nuclear targeting sequence (NTS), similar in structure and function to those found in retroviral regulatory proteins. PTHrP was identified in the nucleus of several different cell types in vivo and in vitro, where it has been implicated in cell cycle progression, cellular differentiation, and apoptosis. In previous work we showed that nuclear translocation of PTHrP enhanced the survival of serum-deprived chondrogenic cells, associated with RNA, and localized to a region of the nucleus rich in complexes of newly transcribed ribosomal RNA and protein. In this work we have used two chondrogenic cell lines, CFK2 (PTH1R+) and 27m21 (PTH1R-) to further explore mechanisms whereby PTHrP rescues immature chondrocytes from apoptosis. Endogenous PTHrP and exogenous PTHrP NTS peptide protected serum-deprived cells from apoptosis, in the presence and absence of PTH1R. The survival of cells expressing PTHrP and those treated with PTHrP NTS peptide was associated with a rapid shift into G(o)/G1 accompanied by a significant down-regulation of rRNA synthesis and a decrease in the number of actively translating polyribosome complexes. Together with our previous observations, this work predicts a role for PTHrP in modulating ribosome biogenesis and preventing chondrogenic cells from progressing through the cell cycle in an unfavorable environment.

Base Sequence↗

PubMed2001

Disruption of NF-kappa B signaling and chemokine gene activation by retroviral mediated expression of IKK gamma/NEMO mutants.

Phosphorylation of I kappa Bs--the cytoplasmic inhibitors of the NF-kappa B transcription factors--is the key event which triggers activation of the NF-kappa B cascade. Signal-mediated phosphorylation of I kappa B alpha is mediated by a multiprotein complex, the I kappa B kinase (IKK) complex, which is composed of at least three identified subunits. Two of these polypeptides, IKK alpha and IKK beta, also known as IKK1 and IKK2, are the catalytic subunits of the kinase complex and phosphorylate I kappa B alpha and I kappa B beta. The third component, NEMO/IKK gamma, does not exhibit kinase activity, but rather constitutes a regulatory subunit. In the present study, C-terminal truncated forms of IKK gamma--Delta C-IKK gamma 306 and Delta C-IKK gamma 261--were stably expressed in the myeloid cell line U937 by retroviral-mediated gene transfer. Overexpression of Delta C-IKK gamma resulted in a reduction in IKK kinase activity in vitro, a subsequent decrease in NF-kappa B DNA binding activity, and inhibition of chemokine gene induction in response to TNFalpha stimulation or paramyxovirus infection. This study demonstrates the efficacy of Delta C-IKK gamma as a repressor of IKK signaling and NF-kappa B activation and suggests a potential gene therapy approach to limit chronic inflammation due to chemokine hyperactivation.

Blotting, Western↗

PubMed2001

Transcriptional regulation of the TFIIH transcription repair components XPB and XPD by the hepatitis B virus x protein in liver cells and transgenic liver tissue.

Human hepatitis B virus is a risk factor for the development of hepatocellular carcinoma. The hepatitis B virus x protein (HBx) has been shown to inactivate the p53 tumor suppressor protein and impair DNA repair, cell cycle, and apoptosis mechanisms. Herein we report that HBx represses two components of the transcription-repair factor TFIIH, XPB (p89), and XPD (p80), both in p53-proficient and p53-deficient liver cells. This inhibition is observed while HBx maintains its transactivation function. Expression of HBx in liver cells results in down-regulation of endogenous XPB and XPD mRNAs and proteins; this inhibition is not observed with other TFIIH subunits, XPA or PCNA. In liver tissue from HBx transgenics, XPB and XPD proteins are down-regulated in comparison to matched normal liver tissue. HBx has been shown to interact with Sp1 transcription factor and affects its DNA binding activity. Sp1 is essential for the basal promoter activity of XPB in liver cells and Drosophila SL2 cells. In the Sp1-deficient SL2 cells, HBx-induced XPB and XPD inhibition is Sp1-dependent. In summary, our results provide evidence that HBx represses the expression of key TFIIH proteins at least in part through Sp1 elements; this repression may impair TFIIH function in DNA repair mechanisms.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

Pseudotyped retrovectors for tumor-specific delivery of toxic suicide genes.

Tumor-targeted gene transfer of the suicide gene, herpes simplex virus thymidine kinase (HSV-TK) is an extremely powerful biopharmaceutical approach for the treatment of cancer. However, no substantive clinical benefit has been reported since this concept was first developed more than a decade ago. This review summarizes the current status of human clinical trials employing viral-vector based delivery of HSV-TK as well as novel means being developed by which killing of tumor cells could be enhanced. In particular, we discuss of the use of VSV-G pseudotyped retrovectors in successfully achieving high tumor-restricted gene transfer efficiency in preclinical studies and the challenges still to be overcome to bring cancer gene therapy safely to the clinic.

Journal Article↗

PubMed2000

Neuronal differentiation and growth control of neuro-2a cells after retroviral gene delivery of connexin43.

Given the roles proposed for gap junctional intercellular communication in neuronal differentiation and growth control, we examined the effects of connexin43 (Cx43) expression in a neuroblastoma cell line. A vesicular stomatitis virus G protein (VSVG)-pseudotyped retrovector was engineered to co-express the green fluorescent protein (GFP) and Cx43 in the communication-deficient neuro-2a (N2a) cell line. The 293 GPG packaging cell line was used to produce VSVG-pseudotyped retrovectors coding for GFP, Cx43, or chimeric Cx43.GFP fusion protein. The titer of viral supernatant, as measured by flow cytometry for GFP fluorescence, was approximately 2.0 x 10(7) colony form units (CFU)/ml and was free of replication-competent retroviruses. After a 7-day treatment with retinoic acid (20 microm), N2a transformants (N2a-Cx43 and N2a-Cx43.GFP) maintained the expression of Cx43 and Cx43.GFP. Expression of both constructs resulted in functional coupling, as evidenced by electrophysiological and dye-injection analysis. Suppression of cell growth correlated with expression of both Cx43 or Cx43.GFP and retinoic acid treatment. Based on morphology and immunocytochemistry for neurofilament, no difference was observed in the differentiation of N2a cells compared with cells expressing Cx43 constructs. In conclusion, constitutive expression of Cx43 in N2a cells does not alter retinoic acid-induced neuronal differentiation but does enhance growth inhibition.

Cell Differentiation↗

PubMed2000

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.

Animals↗

PubMed2000

Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis.

Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8, 9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2000

Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages.

OBJECTIVES: Marrow stromal cells are mesenchymal stem cells able to differentiate into cardiomyocytes in vitro. We tested the hypothesis that marrow stromal cells, when implanted into myocardium, can undergo milieu-dependent differentiation and express cardiomyogenic phenotypes in vivo. METHODS: Isogenic adult rats were used as donors and recipients to simulate autologous transplantation. Marrow stromal cells isolated from donor leg bones were culture-expanded, labeled with 4;,6-diamidino-2-phenylindole, and then injected into the myocardium of the recipients. The hearts were harvested from 4 days to 12 weeks after implantation, and the implant sites were examined to identify the phenotypes of the labeled marrow stromal cells. RESULTS: Viable cells labeled with 4;, 6-diamidino-2-phenylindole can be identified in host myocardium at all time points after implantation. Implanted marrow stromal cells show the growth potential in a myocardial environment. After 4 weeks, donor cells derived from marrow stromal cells demonstrate myogenic differentiation with the expression of sarcomeric myosin heavy chain and organized contractile proteins. Positive staining for connexin 43 indicates the formation of gap junctions, which suggests that cells derived from marrow stromal cells, as well as native cardiomyocytes, are connected by intercalated disks. CONCLUSIONS: Different cell sources have been used as donor cells for cellular cardiomyoplasty. Our findings indicate that marrow stromal cells can also be used as donor cells. In an appropriate microenvironment they will exhibit cardiomyogenic phenotypes and may replace native cardiomyocytes lost by necrosis or apoptosis. Because marrow stromal cells can be obtained repeatedly by bone marrow aspiration and expanded vastly in vitro before being implanted or used as autologous implants, and because their use does not call for immunosuppression, the clinical use of marrow stromal cells for cellular cardiomyoplasty appears to be most advantageous.

Animals↗

PubMed1999

Vesicular stomatitis virus G pseudotyped retrovector mediates effective in vivo suicide gene delivery in experimental brain cancer.

Direct in vivo tumor-targeting with "suicide" viral vectors is limited by either inefficient gene transfer (i.e., retroviral vectors) or indiscriminate transfer of a conditionally toxic gene to surrounding nonmalignant tissue (i.e., adenoviral vectors). Retrovectors pseudotyped with the vesicular stomatitis virus G protein (VSVG) may serve as a remedy to this conundrum. These retroviral particles differ from standard murine retroviruses by their very broad tropism and the capacity to be concentrated by ultracentrifugation without loss of activity. We propose that a VSVG-typed retrovector can be used for efficient and tumor-specific herpes simplex virus thymidine kinase (TK) gene delivery in vivo. To test this hypothesis, we developed a bicistronic retroviral vector that expresses TK and green fluorescence protein (pTKiGFP). The 293GPG packaging cell line was used to generate vTKiGFP retroparticles. In cytotoxicity assays, vTKiGFP-transduced human glioma cell lines were sensitized to the cytotoxic effects of gangciclovir (GCV) 10,000-fold. Subsequently, virus was concentrated by ultracentrifugation to a titer of 2.3 x 10(10) cfu/ml. We tested the antitumor activity of vTKiGFP retroparticles in a rat C6 glioma model of brain cancer. Concentrated retrovector stock (9 microl volume) was injected stereotactically in preestablished intracerebral tumor. Subsequently, rats were treated with GCV for 10 days. Control rats (no GCV) had a mean survival of 38 days (range, 20-52 days). Sections performed on postmortem brain tissue revealed large tumors with evidence of high efficiency retrovector transfer and expression (as assessed by GFP fluorescence). Fluorescence was restricted to malignant tissue. In the experimental group (GCV treated), 8 of 12 remain alive and well >120 days after glioma implantation. In conclusion, vTKiGFP is very efficient at transducing human glioma cell lines in vitro and leads to significant GCV sensitization. Recombinant retroviral particles can be concentrated to titers that allow in vivo intratumoral delivery of large viral doses. The therapeutic efficiency of this reagent has been demonstrated in a preclinical model of brain cancer.

Animals↗

PubMed1999

Effects of retroviral-mediated MDR1 expression on hematopoietic stem cell self-renewal and differentiation in culture.

Ex vivo expansion of hematopoietic stem cells would be useful for bone marrow transplantation and gene therapy applications. Toward this goal, we have investigated whether retrovirally-transduced murine stem cells could be expanded in culture with hematopoietic cytokines. Bone marrow cells were transduced with retroviral vectors expressing either the human multidrug resistance 1 gene (HaMDR1), a variant of human dihydrofolate reductase (HaDHFR), or both MDR1 and DHFR in an internal ribosomal entry site (IRES)-containing bicistronic vector (HaMID). Cells were then expanded for 15 days in cultures stimulated with interleukin (IL)-3, IL-6, and stem cell factor. When very low marrow volumes were injected into lethally irradiated recipient mice, long-term reconstitution with 100% donor cells was seen in all mice injected with HaMDR1- or HaMID-transduced cells. By contrast, engraftment with HaDHFR- or mock-transduced cells ranged from partial to undetectable despite injection of significantly larger marrow volumes. In addition, mice transplanted with expanded HaMDR1- or HaMID-transduced stem cells developed a myeloproliferative disorder that was characterized by an increase in abnormal peripheral blood leukocytes. These results show that MDR1-transduced stem cells can be expanded in vitro with hematopoietic cytokines, but indicate that an increased stem cell division frequency can lead to stem cell damage.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Cyclic-AMP induction of gap junctional intercellular communication increases bystander effect in suicide gene therapy.

The phenomenon of the "bystander effect" (BE) observed in suicide gene therapy studies leads to the intriguing possibility that cytotoxicity can be achieved even in tumor cells that have not themselves been targeted with novel genetic material. There is considerable data suggesting the role of gap junction-mediated intercellular communication (GJIC) in the BE. Transfer of connexin (Cx)-encoding genes, the building blocks of GJIC, has been shown both in vitro and in vivo to increase the BE. Since the loss of GJIC is a common feature of cancer cells, we examined the consequence of GJIC up-regulation on the BE in suicide gene therapy. We used 8-bromo-cyclic-AMP to induce Cx43 and GJIC. In mixing assays, using various proportions of cells containing viral thymidine kinase delivered by an adenoviral delivery system or stably transduced by a retrovirus vector, 8-bromo-cyclic-AMP enhanced the BE of cell killing using ganciclovir. The induction in cell killing was more significant when a low percentage of the cell population was infected, which is the relevant clinical situation. We have demonstrated that this is not due to an effect on infectivity or suicide gene expression. Since decreased GJIC is part of the transformed phenotype, induction of Cxs provides an element of selectivity to suicide gene therapy. Our study adds strength to the rationale to develop clinically tolerable GJ inducers to potentiate the effect of suicide gene therapy via the BE.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Transduction of murine bone marrow cells with an MDR1 vector enables ex vivo stem cell expansion, but these expanded grafts cause a myeloproliferative syndrome in transplanted mice.

Attempts to expand repopulating hematopoietic cells ex vivo have yielded only modest amplification in stem cell numbers. We now report that expression of an exogenous human multi-drug resistance 1 (MDR1) gene enables dramatic ex vivo stem cell expansion in the presence of early acting hematopoietic cytokines. Bone marrow cells were transduced with retroviral vectors expressing either the MDR1 gene or a variant of human dihydrofolate reductase (DHFR), and then expanded for 12 days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor. When these cells were injected into nonirradiated mice, high levels of long-term engraftment were only seen with MDR1-transduced grafts. To verify that expansion of MDR1-transduced repopulating cells had occurred, competitive repopulation assays were performed using MDR1 expanded grafts. These experiments showed progressive expansion of MDR1-transduced repopulating cells over the expansion period, with a 13-fold overall increase in stem cells after 12 days. In all of the experiments, mice transplanted with expanded MDR1-transduced stem cells developed a myeloproliferative disorder characterized by high peripheral white blood cell counts and splenomegaly. These results show that MDR1-transduced stem cells can be expanded in vitro using hematopoietic cytokines without any drug selection, but enforced stem cell self-renewal divisions can have adverse consequences.

ATP Binding Cassette Transporter, Subfamily B, Mem↗