Takahito Yazaki

Intravesical and intravenous therapy of human bladder cancer by the herpes vector G207.

G207, a conditionally replicating herpes vector, efficiently kills human bladder cancer cells in vitro. To evaluate the therapeutic potential of G207, we have established three in vivo models similar to the clinical situation. In vivo, G207 was intraneoplastically, intravesically, or intravenously inoculated in nude mice. Intraneoplastic inoculation into subcutaneous tumor caused significant tumor growth inhibition. Intravesical inoculation of G207 also caused decreased tumor growth in an orthotopic human bladder cancer model. Furthermore, multiple intravenous inoculation markedly inhibited subcutaneous tumor growth. These results suggest that intravesical therapy with G207 is effective for localized bladder tumor, especially for carcinoma in situ (CIS), and intravenous therapy with G207 is promising for invasive or metastasized bladder tumor.

Glycopeptide of P0 protein inhibits homophilic cell adhesion. Competition assay with transformants and peptides.

Expression of major myelin glycoprotein P0 by PO cDNA transfection into C6 glioma cells promoted homophilic cell adhesion of the cells. After the dissociated cells were incubated for various times, the number of particles at each time point was measured. The total number of particles decreased to 24% in 60 min for transformant (C6P0) cells, in contrast to only 68% for control (C6P0′) cells. To confirm the homophilic mechanism of adhesion, mixed‐cell aggregation experiments were performed. Among the four synthetic peptides corresponding to a part of the P0 sequence used, only peptide 3 (residues 90–96), which contained a carbohydrate attaching site, caused considerable inhibition of cell aggregation (approximately 50%). In addition, the glycopeptide (residues 91–95) obtained from bovine P0 markedly inhibited cell aggregation (by approximately 85%).

Expression of L1 and TAG‐1 in the corticospinal, callosal, and hippocampal commissural neurons in the developing rat telencephalon as revealed by retrograde and in situ hybridization double labeling

In the telencephalon, the corticospinal (CS), callosal, and hippocampal commissural neurons are the major types of neurons that have axons crossing the midline of the brain. To understand the mechanisms involved in crossing the midline structure and to examine whether the expression patterns of L1 and TAG‐1 in the commissural neurons are similar to those in the spinal cord, we investigated L1 and TAG‐1 expression in these neurons in rats by using a double‐labeling technique involving retrograde labeling and in situ hybridization. Expression of L1 messenger RNA was detected in the retrogradely labeled CS projection neurons by 1,1`‐dioctadecyl‐3,3,3`,3`‐tetramethylindocarbocyanine perchlorate (DiI) injection into the pons at embryonic day (E) 19, but expression of TAG‐1 messenger RNA was not detected in these neurons. Also, after their axons crossed the pyramidal decussation, continued expression of L1 but no expression of TAG‐1 in the CS projection neurons was shown by an additional double‐labeling experiment involving DiI injection into the spinal cord at postnatal day (P) 1. An immunohistochemical study showed that L1 was continuously present in each level of the CS tract at E21 and P3, but TAG‐1 immunoreactivity was not found in any level at any stage. Finally, we examined the expression of L1 and TAG‐1 messenger RNAs in the callosal and hippocampal commissure neurons after their axons had crossed the midline by using the double‐labeling technique. In both cases, hybridization signals of the L1 and TAG‐1 messenger RNAs were observed in the retrogradely labeled neurons at P3. These results suggest that the roles of L1 and TAG‐1 in the formation of the commissures in the forebrain are different from their roles in the spinal cord. J. Comp. Neurol. 417:275–288, 2000.

Antitumoral effects of defective herpes simplex virus-mediated transfer of tissue inhibitor of metalloproteinases-2 gene in malignant glioma U87 in vitro : Consequences for anti-cancer gene therapy

We set up experiments to evaluate the effects of defective herpes simplex virus (HSV)-mediated in vitro gene transfer of tissue inhibitor of metalloproteinases-2 (TIMP-2) in malignant glioma cells. Intrinsic TIMPs are known to be inhibitors of the strong invasive activities of matrix metalloproteinases in malignant gliomas. The defective HSV vectors dvSRaTIMP2 was engineered to express human TIMP-2 (hTIMP-2) with a combination of replication-competent HSV mutant, temperature-sensitive HSV-tsK, and amplicon plasmid-containing hTIMP-2. The hTIMP-2 gene was driven by the simian virus 40 promoter. The helper virus (HSV-tsK) was thermosensitive; consequently, this vector could proliferate only at 31.5°C. After infection of U87 human glioblastoma cells with the vector in vitro, expression of TIMP-2 was confirmed by reverse zymography. The U87 cells infected in vitro either with dvSRaTIMP2 or HSV-tsK were efficiently destroyed under replication-permissive conditions (at 31.5°C) and significantly lowered under replication-nonpermissive conditions (at 37°C). The invasive activity of U87 was clearly inhibited by dvSRaTIMP2 infection at both 31.5°C and 37°C. Our studies suggest that TIMP-2 expressing the defective HSV vector is possibly useful for the treatment of malignant brain tumors.

Oncolytic viral therapy for human prostate cancer by conditionally replicating herpes simplex virus 1 vector G207.

Over the last few years, a conditionally replicating herpes simplex virus 1 (HSV‐1) vector, G207 has been used for the treatment of several malignant tumors. In this article we evaluate the antitumoral effect of G207 against prostate cancer in vitro and in vivo. The susceptibility of the human prostate cancer cell lines, DU145 and PC3 to G207 at a multiplicity of infection (MOI) of 0.1 was examined. In addition, the growth characteristics of G207 were assessed. Athymic mice with s.c. tumors were inoculated in vivo intraneoplastically with 1x107 plaque‐forming units (PFU) of G207. For the pathological analyses, s.c. tumors were stained with X‐gal. DU145 and PC3 were efficiently destroyed by G207 within 7 days. The viral yields of G207 increased time‐dependently. In vivo, the intraneoplastic inoculation of G207 induced a significant inhibition of the tumor growth. The mean tumor growth ratio was significantly inhibited in the G207‐treated tumors (DU145, P< 0.0001; PC3, P < 0.001 versus controls). In a pathological study, many lacZ‐positive cells were diffusely present in the G207‐treated tumors. G207 showed a significant antitumoral effect against human prostate cancer cell lines, and thus may be considered a useful agent for the treatment of prostate cancer.

Enhanced therapeutic efficacy of oncolytic herpes vector G207 against human non-small cell lung cancer—expression of an RNA-binding protein, Musashi1, as a marker for the tailored gene therapy

Oncolytic herpes vectors like G207 have shown considerable promise in the treatment of solid tumors, but their potency must be enhanced for the full achievement of therapeutic efficacy. Deletion of the innate γ34.5 gene made these vectors extremely safe, but their efficacy was also severely attenuated. Use of tumor‐specific promoters is one method to direct toxicity and enhance efficacy against tumors. Recently, Musashi1 has been shown expressed in some tumor tissues.

Peripheral myelin P0 protein mediates neurite outgrowth of cortical neurons in vitro and axonal regeneration in vivo

Peripheral myelin P0 protein is a homophilic adhesion molecule of immunoglobulin superfamily to compact myelin structure. In addition to its roles in formation and maintenance of myelin, P0 shows neurite-outgrowth promotion activity of dorsal root ganglions. In this paper, we examined biological activity of P0 in central nervous system (CNS). Neurite outgrowth of cortical neurons of rat embryo was markedly promoted in the co-culture on C6 transformants expressing P0 protein. The neurite outgrowth was not inhibited by the P0-glycopeptide but specifically inhibited by the anti-P0 monoclonal antibody recognizing the extracellular peptide of P0. In in vivo studies, we observed significant axonal regeneration into grafts only in animals implanted with P0-expressing transformants after spinal one-third transection. These results suggest that P0 protein has promoting activity on the neurite elongation in CNS as well as in peripheral nervous system.

Multiple isoforms of neuregulin are expressed in developing rat dorsal root ganglia

Accumulating evidence suggests that neuregulin (NRG) plays special roles in the development of the mammalian nervous system. We have already identified NRG as a survival factor for Schwann cells during development. In this report, we have studied all possible NRG isoforms and expression of NRG in the developing rat dorsal root ganglia (DRG) and compared them with those of brain and spinal cord. Neural NRG isoforms comprise common immunoglobulin and epidermal growth factor domains. Various different transcripts were characterized, which arose by alternative splicing in several regions: N‐terminal (exon 1 or 2), spacer (exon 5), juxtamembrane (exon 9 or 10), and cytoplasmic (exon 12, 13, or 14) domains. At least 13 novel isoforms among 16 splice variants were identified. The transmembrane isoforms of NRG are dominant forms in developing rat DRG. The mRNA expression of NRG isoforms in DRG is similar to that in spinal cord, while in brain the expression is much less. The mRNA in DRG was found at similar levels from birth to postnatal day 7 of the premyelinating stage, and it decreased afterward. Our results suggest that several NRGs, including isoforms not reported before, play a role as survival factors for Schwann cells in the premyelinating stage. J. Neurosci. Res. 50:673–683, 1997. © 1997 Wiley‐Liss, Inc.

TREATMENT OF HUMAN RENAL CELL CARCINOMA BY A CONDITIONALLY REPLICATING HERPES VECTOR G207

PURPOSE Surgical removal remains the only potentially curative therapy for renal cell carcinoma. In this study we evaluated the inhibitory effect of the replication competent engineered herpes simplex virus type 1, G207, for renal cell carcinoma in vitro and in vivo. MATERIALS AND METHODS The nature of G207 enables it to replicate within cancer cells, thus, causing cytolysis, but replication is restricted within normal cells. The susceptibility of the human renal cancer cell lines ACHN and A498 to G207 at a multiplicity of infection of 0.1 was examined. In addition, the growth characteristics of G207 was assessed. In vivo athymic mice bearing subcutaneous tumors were inoculated with 1 x 10(7) plaque forming units of G207 intra-neoplastically. For pathological analysis subcutaneous tumors were stained with X-gal. RESULTS Two cell lines were efficiently destroyed by G207 within 1 week. The viral yields of G207 increased in a time dependent manner. In vivo the intra-neoplastic inoculation of G207 caused significantly decreased tumor growth in athymic mice harboring subcutaneous human renal cancer cells. On day 14 the mean growth ratio of ACHN and A498 lesions was significantly inhibited in G207 treated compared to control tumors (p <0.005 and <0.0001, respectively). CONCLUSIONS These results suggest that G207 should be considered another potential therapeutic agent for renal cell carcinoma.

Decrease of NCAM expression and astrocyte-neurone interaction in long-term cultured astrocytes.

&NA; In order to assess the characteristics of the older astrocyte, we obtained long‐term cultured rat astrocytes (20 months) and examined the features of protein expression in relation to neuronal interaction. In short‐term cultured astrocytes, NCAM expressed strongly in contrast to weak expression of laminin by both immunocytochemical and ELISA assay. On the contrary, in long‐term cultured astrocytes, a marked decrease of NCAM expression was observed along with increased laminin expression compared with short‐term cultured astrocytes. The long‐term cultured astrocytes remained positive to anti‐GFAP antibody and showed a much lower ability to interact with neurones than the short‐term cultured astrocytes. NCAM may be one of the responsible molecules related to the astrocyte‐neurone interaction in the developing and ageing nervous system.