I kappa B-mediated apoptotic gene therapy against acute myelogenous leukemia using replication-defective HSV-1 vector expressing TK and mutant I kappa B alpha.

Overexpression of NF-kappa B reportedly plays anti-apoptotic roles in the growth of AML cells. Control of AML cell growth was attempted using a replication-defective herpes simplex virus-1 vector, T0I kappa B alpha, overexpressing mutant I kappa B alpha to inhibit NF-kappa B in vitro. T0I kappa B alpha displays defective ICP4/ICP22/ICP27, isogenic thymidine kinase, and mutant I kappa B alpha. T0Z.1 expressing lacZ instead of I kappa B was used for controls. Infection of T0I kappa B alpha at 15 multiplicity of infection (MOI) with cells of AML lines, HL60, K562, and NB4 displaying >90% infection efficiency and tumor killing in vitro. Use of 10 microM of Ara-C alone was clinically equivalent to high-dose Ara-C, displaying 11% tumor killing. Neither ganciclovir (GCV) nor Ara-C enhanced T0I kappa B- alpha mediated tumor killing. Attenuation of NF-kappa B by T0I kappa B alpha was confirmed by EMSA. T0I kappa B alpha induced caspase-3 activity, with subsequent apoptosis confirmed by colorimetric and TUNEL assays. Fresh AML cells from 8 patients were infected with T0I kappa B alpha at 3 MOI, with or without GCV or 10 microM of Ara-C in vitro. Infection efficiency was 10%. T0I kappa B alpha displayed 8-15% tumor killing, superior to Ara-C in 6 of the 8 patients. Administration of Ara-C enhanced tumor killing in 5 of these 6 cases. Our results suggest that T0I kappa B alpha-mediated gene therapy induces apoptosis of AML cells in vitro.

Sustained release of low-dose ganciclovir from a silicone formulation prolonged the survival of rats with gliosarcomas under herpes simplex virus thymidine kinase suicide gene therapy.

A silicone formulation of ganciclovir (GCV-pellet) was developed to enhance the cytotoxic effects of herpes simplex virus thymidine kinase suicide gene therapy. The effectiveness of this drug delivery system was assessed in a rat 9L gliosarcoma model. The GCV-pellets (1 mm in length and in diameter) used in this experiment contained a total amount of 0.15 mg of GCV. In vitro experiments demonstrated that GCV was gradually released over a period of 7 days. Five days after stereotactic tumor inoculation into the right caudate nucleus, a herpes simplex virus type 1 (HSV-1) vector expressing herpes simplex virus thymidine kinase (HSV-tk) (T1, 2x10(6) pfu) was administered at the same location. The survival rate of the group treated with the GCV-pellet was compared with that of the T1 group injected intraperitoneally (IP) with GCV (30 mg/kg/day for 7 days). The GCV-pellet-treated group had a significantly prolonged survival (a median of more than 80 days) compared with the GCV IP group (a median of 65 days) and with control groups (P<0.05). The control groups (untreated or receiving only the virus vector) had a survival of 35-38 days. The survival rate of the GCV-pellet group over 80 days was 75%, and all the rats that survived more than 80 days and did not show tumors upon histological examination of the brain were deemed cured. No toxic effects or immunological reactions were observed histologically around the pellet in brain sections from the rats treated with the GCV-pellet. After GCV-pellet inoculation into the tumor, drug concentrations were kept at 1-10 microg/g tissue for 3-4 days. When the same dose of GCV (0.15 mg) in aqueous solution was injected into the tumor, GCV concentrations reached a peak of 0.5 mg/g tissue after 30 min and decreased below measurable level within 12 h. After IP injections of 3 mg GCV, GCV concentrations in the tumor reached a peak of 5.7 microg/g tissue after 30 min and also decreased below measurable level within 12 h. This sustained release of a low and effective GCV dose with the silicone formulation significantly prolonged survival in combinations with HSV-tk expression if compared to IP administration of GCV. Histological examination suggests that the treatment appears to be safe.

Double suicide gene therapy using a replication defective herpes simplex virus vector reveals reciprocal interference in a malignant glioma model.

Herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli cytosine deaminase (CD) are non-mammalian enzymes capable of converting innocuous prodrugs into cytotoxic metabolites. Both enzymes have been utilized independently, as well as together in 'suicide' gene therapy protocols to eliminate tumor cells in vitro and in vivo. We have used a set of replication defective HSV vectors expressing either or both enzymes to compare the efficacies of single and double suicide gene therapies in the 9L gliosarcoma model in vitro and in vivo. In cell culture experiments at high and low multiplicities of infection, combined expression of the two genes by vector TOCD/TK along with exposure to the matching prodrugs (ganciclovir and 5-fluorocytosine) showed increased cytotoxicity compared with exposure to either prodrug alone. However, the two gene combination was inferior to single gene treatments, suggesting that HSVtk and CD are mutually counteractive in the prodrug-dependent killing of glioma cells. In animal experiments, survival was not significantly prolonged by administration of both prodrugs to TOCD/TK-treated animals, while each single gene/prodrug pair resulted in increased survival. These results indicate that single suicide gene systems employing HSVtk or CD may be preferable over combinations of the two.

Latent prolactinoma on MRI-selective venous sampling and trans-sphenoidal microsurgical treatment.

Bilateral and simultaneous selective venous sampling from the cavernous sinus, inferior petrosal sinus, jugular vein and peripheral vein was performed in 13 patients with hyperprolactinemia in whom dynamic magnetic resonance imaging failed to reveal pituitary adenoma. The prolactin level in peripheral veins of the patients on admission ranged from 35 to 141 ng ml(-1), with a mean value of 69 ng ml(-1). All patients showed disturbance of menstruation or galactorrhea. The indication for surgery in the present study was extremely rare; the patients each wanted to become pregnant and had an intolerance of dopamine agonists. Trans-sphenoidal surgery was performed based on the results of selective venous sampling. The postoperative levels of prolactin were normalized in nine of the patients and normal pituitary function was preserved after surgery. The present study revealed a correspondence of laterality of the peak prolactin level with the main tumor location in patients with latent prolactinoma. However, the tumor/nontumor ratio did not necessarily coincide with the pattern of tumor location, and tumor location was accurately predicted in only 70% of the cases. Selective venous sampling directly from central veins is useful for diagnosis of microprolactinoma.

HSV vector cytotoxicity is inversely correlated with effective TK/GCV suicide gene therapy of rat gliosarcoma.

Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (tk) gene to tumor cells in combination with ganciclovir (GCV) administration may provide an effective suicide gene therapy for destruction of malignant glioblastomas. However, because HSV is a highly cytotoxic agent, gene expression from the virus is short-lived which may limit the effectiveness of HSVtk/GCV therapy. Using different replication-defective HSVtk gene vectors, we compared HSV vector backgrounds for their cytotoxic activity on infection of 9L gliosarcoma cells in culture and brain tumors in rats and evaluated the impact of vector toxicity on the effectiveness of tk/GCV-mediated suicide gene therapy. As reported previously for other cell lines, a vector deleted for both copies of the immediate-early (IE) gene ICP4 (SOZ.1) was highly toxic for 9L cells in culture while a vector deleted in addition for the ICP22 and ICP27 IE genes (T.1) reduced or arrested 9L cell proliferation with more limited cell killing. Nevertheless, both vectors supported widespread killing of uninfected cells in the presence of GCV following low multiplicity infections, indicating that vector cytotoxicity did not preempt the production of vector-encoded TK enzyme necessary for the killing of uninfected cells by the HSV-tk/GCV bystander effect. Although an SOZ.1-related vector (SHZ.2) caused tumor cell necrosis in vivo, injection of SHZ.2 at multiple coordinates thoughout the tumor followed by GCV administration failed to prolong markedly the survival of tumor-bearing rats. In contrast, a single injection of T.1 produced a life-extending response to GCV. These results indicate that vector cytotoxicity can limit the efficacy of HSV-tk/GCV treatment in vivo, which may be due to premature termination of tk gene expression with attendant abortion of the bystander effect.

Effective treatment of experimental glioblastoma by HSV vector-mediated TNF alpha and HSV-tk gene transfer in combination with radiosurgery and ganciclovir administration.

Experiments were carried out in a nude mouse model of human glioblastoma to determine whether gamma-knife radiosurgery combined with herpes simplex virus thymidine kinase (tk) suicide gene therapy and tumor necrosis factor alpha (TNFalpha) gene transfer provided an improved multimodality treatment of this disease. Animals were inoculated intracerebrally with 2 x 10(5) U-87MG human glioblastoma cells to establish brain tumors. At 3 days postinoculation, the tumor region was injected with 2 x 10(6) infectious particles of highly defective herpes simplex viral vectors expressing the viral tk gene with the kinetics of a viral immediate early gene either alone (T.1) or together with TNF alpha (TH:TNF). Subgroups of animals were given daily intraperitoneal injections of ganciclovir (GCV) for 10 days and/or subjected to gamma-knife radiosurgery on the fifth day post tumor-cell implantation. Comparisons of animal survival showed that the TH:TNF vector in combination with radiosurgery and GCV administration provided the most effective therapy; eight of nine animals survived for 75 days compared to four of eight using the next best protocol. These findings suggest that gene therapy in combination with more conventional therapeutic methods may provide an improved strategy for extending the life expectancy of patients afflicted with this ultimately fatal disease.

Connexin 43-enhanced suicide gene therapy using herpesviral vectors.

Tumor cell transduction with the herpes simplex virus (HSV) thymidine kinase (tk) gene and treatment with ganciclovir (GCV) is a widely studied cancer gene therapy. Connexin (Cx)-dependent gap junctions between cells facilitate the intercellular spread of TK-activated GCV, thereby creating a bystander effect that improves tumor cell killing. However, tumor cells often have reduced connexin expression, thus thwarting bystander killing and the effectiveness of TK/GCV gene therapy. To improve the effectiveness of this therapy, we compared an HSV vector (TOCX) expressing Cx43 in addition to TK with an isogenic tk vector (TOZ.1) for their abilities to induce bystander killing of Cx-positive U-87 MG human glioblastoma cells and Cx-negative L929 fibrosarcoma cells in vitro and in vivo. The results showed that low-multiplicity infection of U-87 MG cells with TOCX only minimally increased GCV-mediated cell death compared with infection by TOZ.1, consistent with the endogenous level of Cx in these cells. In contrast, bystander killing of L929 cells was markedly enhanced by vector-mediated expression of Cx. In vivo experiments in which U-87 MG cells were preinfected at low multiplicity and injected into the flanks of nude mice showed complete cures of all animals in the TOCX group following GCV treatment, whereas untreated animals uniformly formed fatal tumors. TOCX injection into U-87 MG intradermal and intracranial tumors resulted in prolonged survival of the host animals in a GCV-dependent manner. Together, these results suggest that the combination of TK and Cx may be beneficial for the treatment of human glioblastoma.

Efficient gene delivery into epstein-barr virus (EBV)-transformed human B cells mediated by replication-defective herpes simplex virus-1 (HSV-1): A gene therapy model for EBV-related B cell malignancy.

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.

Enhanced tumor cell killing in the presence of ganciclovir by herpes simplex virus type 1 vector-directed coexpression of human tumor necrosis factor-alpha and herpes simplex virus thymidine kinase.

Past studies have documented the promise of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) suicide gene therapy as a potential antitumor treatment. HSV-TK converts the pro-drug ganciclovir (GCV) into a toxic nucleotide analogue, the incorporation of which into cellular DNA blocks cell proliferation. In this report, we have examined the hypothesis that the effectiveness of HSV-TK suicide gene therapy can be enhanced by coexpression of the antitumor cytokine human tumor necrosis factor-alpha (TNF-alpha) from the same replication-defective HSV-1 vector. In vitro testing demonstrated that TNF-alpha expression from this vector potentiated the killing of both TNF-alpha-sensitive L929 tumor cells and TNF-alpha-resistant U-87 MG cells in the presence of GCV. Furthermore, treatment of established intradermal L929 tumors in vivo with the TNF-alpha/TK vector and GCV resulted in prolonged animal survival compared with treatment with parental HSV-TK vector in the presence or absence of GCV. Treatment of intracerebral U-87 MG tumors showed a clear benefit of TK therapy, but a significant further increase in survival using the TNF-alpha vector could not be demonstrated. We found that potentiation of cell killing in vitro required intracellular TNF-alpha because purified protein added to the culture medium of cells infected with HSV-TK vector failed to have the same effect. Accordingly, potentiation in vivo should depend on efficient infection, but immunohistochemical analysis indicated that virus administration by U-87 MG intratumoral injection was inadequate, resulting in an estimated <1% infection of all tumor cells. Moreover, the majority of infected tumor cells were localized at the tumor margin. Together, these results suggest that TNF-enhanced tk gene therapy should provide a useful treatment for TNF-alpha-sensitive tumors and perhaps also for TNT-alpha-resistant tumors if vector delivery can be improved to increase the percentage of transduced tumor cells.

A humanized single-chain Fv fragment with high targeting potential against human malignant gliomas.

A humanized ONS-M21 antibody (hM21) against human medulloblastoma and glioma cells was engineered as a single-chain Fv fragment (scFv), and its ability to internalize into tumor cells was evaluated by conjugation with ricin A. The scFv of hM21 (schM21) was easily purified from E.coli by one-step affinity column chromatography. Purified schM21 bound to a medulloblastoma ONS-76 cell with almost equal antigen-binding activity of hM21-Fab fragment. Furthermore, the schM21-ricin A conjugate inhibited the growth of ONS-76 cells, but not that of antigen-negative hepatoma HuH-7 cells, suggesting that the schM21 can be internalized after binding to antigen-positive cells. Thus, schM21 could be expected to act as a novel carrier of diagnostic and therapeutic agents for brain tumors.

[Effects of biochemical modulation chemotherapy with CDDP and 5-FU on metastatic brain tumor from lung cancer].

Fourteen cases of metastatic brain tumors from lung cancer underwent biochemical modulation chemotherapy with daily administration of small doses of CDDP (5 or 10 mg/day) and continuous infusion of 5-FU (300 mg/day) for three tow six weeks. All patients with metastatic brain tumors also underwent a total of 30 Gy of whole brain irradiation therapy. Of eleven patients who had metastatic brain tumors when chemotherapy started, complete and partial responses were shown in two patients each (36% response rate). Moreover, only four of twelve patients with extracranial lesions responded (33% response rate). Abnormal levels of tumor marker were improved in only three of 10 evaluable patients (30% response rate). Side effects of this chemotherapy included various WHO grades of bone marrow suppression as well as nausea and vomiting in 13 of 14 patients. Loss of appetite persisted for two months. Parkinsonism was also noted in three patients as an additional side effect. Mean survival time was 9.4 months, while 4 patients survived longer than one year. Data showing a low response rate, persistent loss of appetite and longer admission period were considered less favorable than those of other chemotherapeutic regimens.

[Treatment of metastatic brain tumors from lung cancer: analysis of performance status between treatment methods].

Retrospective analysis was performed in 280 patients; 112 surgical cases and 168 non-surgical cases to determine which of three treatments, alone or in combination provides more prolonged improvement of performance status (PS: Karnofsky score) in patients with metastatic brain tumors from lung cancer. The treatments under scrutiny were surgical removal of metastatic brain tumor (S), radiation therapy (R) and chemotherapy (C). KS in the group treated with S or C showed a significantly better result than that in the non-S group or non-C group during the two-year observation period. However, R group showed no significant improvement in KS compared to that in the non-C group during the two-year period. During the first year after admission, two subgroups of S plus R and R alone showed most rapid decrease in KS. However, subgroups of S plus C and S plus R and C showed better results than other subgroups. In analyzing the changes in KS over the short period from admission to one month after treatment had been completed, the non-S group showed a significant decrease in KS, while the S group showed a slight increase in mean KS. The subgroup of R alone showed the greatest decrease in KS. Thus, retrospective analysis showed that surgical removal of a metastatic brain tumor led to improved KS for a short period and chemotherapy was useful in prolonging the duration of better Q.O.L.

Decreased N-myc expression in human medulloblastoma cell lines during differentiation.

Medulloblastoma is the most common primitive neuroectodermal tumor of the central nervous system, and shares some biochemical and immunological features with neuroblastoma. It could be suggested that N-myc expression in medulloblastoma is correlated with primitiveness or cell differentiation, as in neuroblastoma. N-myc expression in two human medulloblastoma cell lines (ONS-76 and -81) was investigated during drug-induced differentiation by immunocytochemistry and Western blotting. The growth rates of the two medulloblastoma cell lines showed a marked decrease and morphological differentiation occurred after treatment with 1 mM dibutyryl cyclic-adenosine 3',5'-monophosphate (dbt-cAMP). The decrease in N-myc expression with differentiation of cells was confirmed by immunocytochemistry and Western blotting. We believe that decreased N-myc expression is correlated with cell differentiation, and a decrease in the primitiveness of medulloblastoma cells.

Hemifacial spasm due to compression of the facial nerve by vertebral artery-posterior inferior cerebellar artery aneurysm and elongated vertebral artery--case report.

A 62-year-old female presented with rapid aggravation of long-standing facial spasm occurring within a few months. Neuroimaging and angiography demonstrated compression of the root exit zone (REZ) of the facial nerve by an ipsilateral saccular aneurysm at the left vertebral artery (VA)-posterior inferior cerebellar artery bifurcation, in addition to the elongated VA. Neck clipping of the aneurysm and decompression of the REZ from the elongated VA and clipped aneurysm resulted in complete disappearance of the facial spasm. The aneurysm had very thin walls and was apparently about to rupture. Aggravation of long-standing hemifacial spasm may be a warning sign for rapid growth and rupture of a causative aneurysm.

Intramedullary spinal cord meningioma--a case report.

A 54-year-old female presented with a spinal intramedullary meningioma manifesting as extremely slow development of dysesthesia in the extremities. Sagittal T1-weighted resonance imaging showed fusiform enlargement of the cervical spinal cord with an area of low-signal intensity suggestive of syringomyelia extending bidirectionally up to the medulla oblongata and downward from the homogeneously enhanced intramedullary space-occupying lesion. The tumor was confirmed to be intramedullary mass with no dural attachment, and was totally removed en bloc. Histological examination showed transitional type of meningioma. The tumor specimen was positive for epithelial membrane antigen, and included glial fibrillary acidic protein-positive fibers, which were thought to be surrounding neural tissues trapped by the nodularity of the tumor in the growing process.

An immunohistochemical analysis of medulloblastoma and PNET with emphasis on N-myc protein expression.

Medulloblastoma is the most common primitive neuroectodermal tumor (PNET) of the central nervous system. Standard whole neuroaxis radiation prolongs survival, but causes mental retardation and growth disturbance. It is important to find appropriate prognostic indicators for medulloblastoma in children. We assessed the prognostic values of N-myc expression in medulloblastoma. Nineteen medulloblastoma or supratentorial PNET (SPNET) patients (15 males and 4 females) were immunohistochemically investigated for N-myc expression. Sixteen patients were N-myc-positive, and three were N-myc-negative. N-myc-positive patients had a tendency towards a poor outcome (P = 0.1125). N-myc-negative tumors were more differentiated towards glial lineage than N-myc-positive tumors. N-myc-negative and GFAP-positive patients (n = 2) tended to survive N-myc-positive and GFAP-negative patients (n = 13). In medulloblastoma and SPNET patients, N-myc expression may become an appropriate indicator of poor prognosis and primitive cell differentiation.

A novel PNET cell line with melanotic differentiation.

The primitive neuroectodermal tumor (PNET) cell line, ONS-99, was established from a human melanotic neuroectodermal tumor of infancy (MNTI). ONS-99 cells were polygonal adherent cells and transplantable to BALB/c-nu/nu mouse. ONS-99 cells were positive for NSE, HMB-45 (melanoma-associated antigen) and N-myc, but lacked the antigenic features of terminally differentiated neurons or glias. ONS-99 cells had many intermediate filaments in the cytoplasm and tight intercellular junctions with scant cytoplasmic structures by electron microscopy. To date, ONS-99 is the only example of an established PNET cell line with melanotic differentiation.

In vitro assessment for neurotoxicity of antitumor agents before local administration into central nervous system.

In vitro assays for neurotoxicity with the aid of cultured mouse fetal neurons and glial cells were applied to investigate neurotoxicity of recombinant murine interferon-beta (rMuIFN-beta). These data were compared with those for MTX, ADR, and ACNU. The range of concentrations of the drugs used in these experiments spanned their clinically achievable concentrations in patient serum (IFN-beta: 1 x 10(4) IU/ml, MTX: 100 micrograms/ml, ADR: 20 micrograms/ml, ACNU: 20 micrograms/ml). rMuIFN- beta damaged both neurons and glial cells at concentrations of more than 1 x 10(5) IU/ml but did not damage them at 1 x 10(4) IU/ml or less. Microtubule-associated protein 1A (MAP1A) staining was decreased in rMuIFN-beta-treated (more than 1 x 10(5) IU/ml) neutrons. In conclusion, since IFN-beta may have some neurotoxic effects at concentrations higher than 1 x 10(5) IU/ml, it should be administered carefully, as should other antitumor agents, into the tumor cavity in the CNS following surgery.