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PubMed1999

Folylpolyglutamyl synthetase gene transfer and glioma antifolate sensitivity in culture and in vivo.

BACKGROUND: Although antifolates are popular agents for use in chemotherapy, they display minimal toxicity against slow-growing tumors and are toxic to actively replicating cells in normal tissues. These drugs are converted intracellularly into polyglutamate derivatives by the enzyme folylpolyglutamyl synthetase (FPGS). Because tumors with high expression of FPGS often respond to nontoxic antifolate doses, we investigated whether augmenting tumoral FPGS activity by gene delivery would enhance tumoral antifolate sensitivity. METHODS: 9L rat gliosarcoma cells were stably transfected with a human FPGS complementary DNA (cDNA), producing 9L/FPGS cells. The sensitivity of these cells to the antifolates methotrexate and edatrexate was measured in culture and in subcutaneous tumors, as was their ability to increase the chemosensitivity of nearby nontransfected cells, i.e., a bystander effect. The antifolate sensitivity of nonselected cells transduced with a hybrid amplicon vector that expressed FPGS was also ascertained. RESULTS: In comparison with 9L cells, 9L/FPGS cells displayed enhanced sensitivity to 4-hour pulses of antifolate. Subcutaneous 9L/FPGS tumors responded as well to methotrexate given every third day as 9L tumors did to daily treatment. A modest bystander effect was observed with edatrexate treatment in culture and in vivo. The observed bystander effect appeared to result from the release of antifolates by transfected cells after the removal of extracellular drug. In culture, enhanced antifolate sensitivity was also seen in other stably transfected rodent and human glioma cell lines, including one with high pre-existing FPGS activity, and in canine and human glioblastoma cell lines transduced with a vector bearing FPGS cDNA. CONCLUSIONS: FPGS gene delivery enhances the antifolate sensitivity of several glioma cell lines and merits further evaluation as a therapeutic strategy.

Aminopterin↗
PubMed1982

Tumor immunity in rat lymph nodes following transplantation.

Inguinal lymph nodes in the Buffalo rat were autotransplanted to the popliteal fossa by an intact vascular pedicle or by microvascular anastomosis. These revascularized nodes had normal histology and made spontaneous afferent and efferent lymphatic reconnection with surrounding lymphatic vessels, as documented by ink and silicone rubber injection studies. Lymphoscintigraphy with 99mTc antimony sulfide colloid correctly predicted the 44 of 120 node transplants that had made afferent reconnection. To demonstrate immunologic activity of lymph nodes following transplantation, a cellular adherance assay was employed to detect cell-mediated cytotoxicity of lymph node cells isolated from rats sensitized to an allogeneic gliosarcoma. Cytotoxicity was detected in nontransplanted regional nodes sensitized to tumor (p less than 0.01) and in nodes transplanted by vascular pedicle and then sensitized to tumor (p less than 0.001). This study demonstrates that lymph nodes can be transplanted with restoration of functional lymphatic anatomy, and that following transplantation, lymph nodes retain the ability to mount an immune response against tumor.

Animals↗
PubMedDate not supplied

Neonatal brain tumors: CT and MR findings.

Twelve neonatal brain tumors, presenting within 60 days of birth, constituted 3.3% of pediatric brain tumors. Three-fourths were supratentorial. Two-thirds were benign. Forty-two percent were choroid plexus papilloma. Twenty-five percent were teratoma. Eight percent each were hypothalamic glioma, gliosarcoma, medulloblastoma, and primitive neuroectodermal tumor. Clinical symptoms were nonspecific. Signs of herniation were absent in all 12 patients. Forty-two percent of these patients died 1 day to 8 months after diagnosis. Ultrasound, CT, and magnetic resonance have all proved useful for displaying these lesions suitably for surgery.

Brain Neoplasms↗
PubMed1983

Immunity to transplantable nitrosourea-induced neurogenic tumors. II. Immunoprophylaxis of tumors of the brain.

An effective method was sought to immunize rats against the growth of intracerebrally (IC) injected T9 tumor, a gliosarcoma cell line. Rats which were immunized with either 10(6) T9 cells mixed with 0.14 mg C. parvum, or 10(7) irradiated T9 cells showed tumor immunity to intradermal (ID) transplantation. However, to obtain tumor immunity to an IC challenge of T9 cells, rats initially had to reject an ID challenge of T9 cells. After sequential rejections of ID challenges of as many as 10(7) cells, a high degree of immunity was obtained, allowing rejection of up to 5 X 10(6) T9 cells injected IC. Spleen cells from highly immunized rats mixed with T9 cells at a ratio of 1:25 (tumor:spleen) destroyed T9 tumor cells in a Winn test. Normal spleen cells had no effect on tumor growth. We conclude that: 1) T9 cells are moderately immunogenic, 2) an effective method of immunization of CDF rats against ID transplanted T9 cells is 10(6) T9 cells with 0.14 mg C. parvum or 10(7) irradiated T9 cells, 3) a higher degree of tumor immunity is necessary to reach the brain than is needed to reach the periphery, and 4) spleen cells of highly immunized rats are cytotoxic to T9 tumor cells.

Animals↗
PubMed1984

Immunity to transplantable nitrosourea-induced neurogenic tumors. III. Systemic adoptive transfer of immunity.

The effect of intravenously injected tumor immune spleen cells on growth of 3 X 10(5) gliosarcoma T9 cells injected intradermally (ID) or intracerebrally (IC) into sublethally irradiated CDF rats was evaluated. Spleen cells from donor rats with sufficient immunity to reject 5 X 10(5) T9 cells inhibited the growth of T9 cells mixed with spleen cells in a ratio of 1:25 and injected ID, but could not act after intravenous transfer. However, donor rats which had rejected increasing T9 challenge doses up to 1 X 10(7) cells produced immune spleen cells which, upon IV transfer, could inhibit growth of ID T9 challenge but not of EB-679, an unrelated glioma, in recipient rats. Rejection of IC T9 challenge was also obtained after IV transfer, in recipients of such "hyperimmune" spleen cells, but was less (60% maximum) than that noted after ID T9 challenge (100% maximum). The removal of B cells from the transferred spleen cells did not affect the results, suggesting that the specific immunity was mediated by T cells. We conclude that the special immunological circumstances of tumors growing in the brain renders them less accessible to rejection by systemically transferred immune cells, but it is nevertheless possible to effect a significant incidence of rejection of syngeneic tumor growth in the brain by the intravenous transfer of hyperimmune spleen cells.

Animals↗
PubMed1988

Gene amplification in malignant human gliomas: clinical and histopathologic aspects.

Gene amplification occurs in 45-50% of malignant human gliomas (MHG). In the present study, 64 genetically characterized gliomas were evaluated to determine if tumors with amplification of the epidermal growth factor receptor (EGFR), N-myc, c-myc, or gli genes had distinctive histopathologic features. There was no significant difference in age (p = 0.10) or gender (p = 0.78) between patients whose tumors contained amplified genes and those whose tumors did not exhibit this characteristic. Although the patients with amplified genes in their tumors survived slightly longer than patients whose tumors had no detectable gene amplification, these differences were not statistically significant (p = 0.21). The 28 tumors with amplification included 24/48 (50%) glioblastoma multiforme, 2/6 (33%) anaplastic astrocytomas and 2/5 (40%) gliosarcomas. No amplification was seen in one oligodendroglioma, three anaplastic mixed gliomas or one giant cell glioblastoma multiforme. Necrosis and endothelial proliferation were equally prevalent among tumors with and without amplification. Comparison of tumors with gene amplification and tumors without this characteristic revealed similar distributions of most morphologic cells types. Although prominent perivascular lymphocytic infiltrates were more frequent in tumors without amplification, this association was of borderline significance statistically. In situ hybridization of tumors with amplification using an EGFR mRNA probe showed intense labeling of different neoplastic cell types including fibrillary and protoplasmic astrocytes, gemistocytes, anaplastic cells, and multinucleated giant cells. Non-neoplastic cells such as hyperplastic endothelium within the tumors did not express detectable EGFR mRNA. These studies demonstrate that (a) cells with quite different morphology within the same tumor can contain the same genetic alteration; (b) tumors of identical histological appearance may have arisen and evolved by different molecular mechanisms; and (c) molecular analyses are necessary to evaluate gene amplification in MHG since this characteristic cannot be accurately predicted by the morphologic or clinical criteria used in this study.

Adult↗
PubMed1991

Occurrence of lacto series gangliosides 3'-isoLM1 and 3',6'-isoLD1 in human gliomas in vitro and in vivo.

Monoclonal antibodies (MAb; DMAb, monoclonal antibodies derived at Duke Medical Center) directed against the oncofetally expressed lactotetraosyl gangliosides 3'-isoLM1 (IV3NeuAc-LcOse4Cer) and 3',6'-isoLD1 (IV3NeuAc,III6NeuAc-LcOse4Cer) were produced and their reactivity spectra compared to that of the alpha-3'-isoLM1 MAb SL-50. The IgM MAb SL-50 defines the epitope NeuAc (or NeuGc)alpha 2-3Gal beta 1-3GlcNAc, the terminal sequence of both gangliosides. SL-50 requires an unsubstituted GlcNAc residue; IgM DMAb-14 will accept the alpha 2-6 linked sialic acid to GlcNAc found in 3',6'-isoLD1. Immunohistochemical localization of 3'-isoLM1 was performed on 31 biopsy specimens of human gliomas; 15 (48%) expressed 3'-isoLM1 as defined by binding of MAb SL-50. Staining of small anaplastic cells, giant cells, and the glial component of gliosarcomas was observed. Neoplastic gemistocytes, when present, showed particularly intense staining. The 3'-isoLM1 and 3',6'-isoLD1 distribution in cultured cell lines and derived xenografts of primary tumors of the human central nervous system and of embryonal or neuroectodermal tumor derivation was determined. Six of 29 cell lines expressed 3'-isoLM1: 2/16 gliomas, 3/3 teratomas, 1/1 pancreatic adenocarcinoma. No cell line expressed detectable 3',6'-isoLD1 by immunostain analysis of ganglioside extracts. The 3'-iso-LM1-positive cell lines expressed it in xenograft form; in five xenografts, the corresponding cell lines of which were 3'-isoLM1-negative, it was a proportion of the monosialoganglioside fraction. 3',6'-isoLD1 was detected in two xenografts, D-54 MG (glioma) and PA-1 (teratoma). The demonstration of 3'-isoLM1 in gliomas in in vivo forms and the relatively infrequent expression by derived cultured cells suggest that ganglioside expression is modified by environmental forces. Expression of 3'-isoLM1 and 3',6'-isoLD1 in fetal and neonatal brain, in intense reactive astrocytosis such as polyunsaturated fatty acid lipidosis, and in primary neoplasms of the central nervous system suggests their role in cell-cell attachment during development, migration, and neoplastic transformation.

Animals↗
PubMed1998

Interleukin-12-based immunotherapy against rat 9L glioma.

OBJECTIVE: Interleukin-12 (IL-12) may be useful for immunotherapy against gliomas because it can reverse the glioma-induced suppression of T-cell proliferation and interferon-gamma production. We postulated that peripheral infusion of IL-12 along with irradiated tumor cells can lead to immunological rejection of 9L glioma. METHODS: 9L gliosarcoma flank tumors were established in syngeneic Fischer 344 rats. Osmotic minipumps delivered IL-12 subcutaneously, and irradiated 9L cells were injected on Days 0, 3, 7, 14, and 21. Tumor volumes were measured by a blinded observer. For tumor rechallenge, animals initially cured of 9L flank tumors received either another implantation of flank tumor or a stereotactic injection of 10(6) 9L cells into the right striatum. Delayed-type hypersensitivity was measured after injecting 10(6) irradiated 9L tumor cells into the right pinnae. RESULTS: Tumor growth curves were significantly different between treated and control animals. Among the animals that received 1 ng per day of IL-12, 40% did not develop any measurable tumors at all. A combination of irradiated 9L cells and IL-12 was necessary for optimal effect. Cured animals rejected future flank tumors. All animals rechallenged with intraparenchymal brain tumors survived, whereas control animals all died by Day 22. Delayed-type hypersensitivity measurements showed a specific and long-lasting response against 9L cells. CONCLUSION: Continuous administration of the lymphokine IL-12, in the presence of irradiated tumor cells for antigen presentation, circumvents the need for gene transfection for generating tumor cell vaccines. We have demonstrated that the combination of IL-12 and irradiated tumor cells can lead to regression of 9L flank tumors and resistance to future flank and central nervous system tumor challenges.

Animals↗
PubMed2002

Hypertonic saline ameliorates cerebral edema associated with experimental brain tumor.

Cerebral edema commonly accompanies brain tumors and frequently leads to lethal intracranial compartmental shifts and elevated intracranial pressure. Therapeutic modalities for tumor-associated cerebral edema include diuretics, osmotherapy, and corticosteroids. Recently, hypertonic saline (HS) has received attention as an osmotic agent in the treatment of cerebral edema from diverse causes. The effects of continuous HS infusion in brain tumor-associated edema have not been previously reported. Therefore, we tested the hypothesis that HS given as a continuous intravenous infusion ameliorates tumor-associated edema in a rat model of brain tumor. 9L gliosarcoma, propagated as a solid flank tumor, was implanted intracranially over the left hemisphere in adult female Fischer 344 rats (180-220 g). On day 11 after implantation, rats were divided in a blinded, randomized fashion into groups that received no treatment or continuous infusion of 0.9% saline (NS) (0.3 mL/h) and in a subsequent series that included NS + intravenous furosemide 2.5 mg/kg every six hours, NS + intravenous mannitol 2.5 g/kg every six hours, or continuous infusion 7.5% HS (chloride:acetate 50:50) (0.3 mL/h). Hemispheric water content ipsilateral (IH) and contralateral to tumor implantation was determined at day 13 by wet-to-dry weight ratio after 48 hours of therapy. Ipsilateral hemispheric water content (mean +/- SEM) was significantly increased in rats with intracranial tumor on day 11 (80.3 +/- 0.5%) (n = 7) and day 13 (81.4 +/- 0.3%) (n = 10), as compared to naive weight-matched rats without tumor implant (79.3 +/- 0.1%) (n = 13) (P <.05). After 48 hours of treatment, IH water content was attenuated with continuous HS (n = 15) (79.3 +/- 0.2%), mannitol (n = 14) (80.1 +/- 0.2%), and furosemide (n = 15) (79.9 +/- 0.2%) as compared to NS (n = 7) (80.8 +/- 0.5%). Continuous HS infusion attenuated cerebral edema in the affected hemisphere as well as the contralateral noninjured hemisphere to a larger extent than was observed with furosemide or mannitol. These findings suggest a potential new treatment strategy for tumor-associated cerebral edema.

Animals↗
PubMed1988

Immunotherapy of solid tumor by intratumoral infusion of lymphokine-activated killer cells.

Fifty million lymphokine-activated killer (LAK) cells were infused into rat T9 gliosarcoma tumors for 1 hr at an infusion rate of 0.1 ml/hr. Cultured normal spleen cells were infused into similar tumors as a control. The LAK cell-treated tumors began to regress at approximately 3 weeks after infusion and disappeared by 6 weeks, while the cultured normal spleen cell-treated tumors grew progressively. Immunohistochemical analysis demonstrated prominent infiltration of cytotoxic/suppressor T cells in the LAK cell-treated tumors, while few lymphocytes were recognized in the control tumors. These data suggested that LAK cells infused intratumorally might be capable of mediating tumor regression by inducing host immunity against the tumor.

Animals↗
PubMed1990

Functional analysis of mononuclear cells infiltrating into tumors: establishment of a new system to obtain functionally active tumor-infiltrating cells.

We have demonstrated that tumor-infiltrating T cells play a crucial role in tumor growth. In order to investigate the precise function of tumor-infiltrating cells, we avoided the use of any enzyme for separation of tumor-infiltrating cells from the solid tumor mass and established a new system in which we could easily obtain functionally active tumor-infiltrating cells in large amounts from the peritoneal cavity of T-9 gliosarcoma-sensitized rats. In this system, viable T-9 cells were intraperitoneally administered and peritoneal exudate cells were recovered. Importantly, cells obtained from the intraperitoneal cavity are phenotypically and functionally indistinguishable from those obtained from subcutaneous tumor masses. Furthermore, injection of purified lymphocyte migration factor-b resulted in the accumulation of CD8 (+) cytotoxic T cells in the peritoneal cavity. Therefore, this system also provides a means by which the in vivo function of chemotactic factor can be tested.

Animals↗
PubMed1981

Effect of tritiated thymidine on the kinetics and viability of 9L cells in vitro.

The colony-forming efficiency of 9L rat gliosarcoma cells was unaffected by treatment with 0.1 muCi/ml of [3H]TdR. However, when cells were treated with 1 or 10 muCi/ml of [3H]Tdr, cell growth was reduced and cell survival decreased. When monolayer 9L cells were treated with 1 muCi/ml of [3H]TdR for up to 72 hr, approximately 5% survived, which is closely related to the percentage of non-cycling cells in this system. When cells were treated with 10 muCi/ml of [3H]TdR for 72 hr, less survival was observed. The additional cell kill observed may be induced by [3H]TdR released from doomed cells into petri dishes during the incubation period of the colony-formation assay.

Animals↗
PubMedDate not supplied

Intracellular serum proteins in cerebral gliomas and metastatic tumours: an immunoperoxidase study.

An immunoperoxidase technique was used to compare the presence of serum proteins in tumour cells in twenty cerebral gliomas and four intracerebral metastatic carcinomas, with the serum protein content of reactive astrocytes around four cerebral infarcts. Rabbit antiserum to human immunoglobulins and albumen was applied to paraffin sections of tumour biopsies and of infarcts from postmortem brains. All the reactive astrocytes surrounding infarcts stained positively for immunoglobulins and albumen. In the gliomas, a continuous range of staining was observed in plump astrocytic tumour cells varying from strongly positive to unstained. Very few poorly-differentiated glioma cells stained for serum proteins; metastatic tumour cells, areas of capillary endothelial proliferation, and spindle cells in gliosarcomas remained unstained. These results suggest that the astrocytic function of serum protein uptake from the extracellular fluid is often lost in poorly-differentiated glioma cells, but is retained to a variable degree by those tumour cells which histologically resemble reactive astrocytes.

Adenocarcinoma↗
PubMed1999

Identification in human brain tumors of DNA sequences specific for SV40 large T antigen.

Simian virus 40 (SV40) sequences have recently been identified in a variety of human neoplasms, including mesothelioma, osteosarcoma, and brain tumors, but significant discrepancies exist regarding the frequency at which this occurs. The SV40 genome is 70% homologous to JC and BK, two related polyomaviruses that are highly prevalent in humans and which may cause in immune-compromised patients progressive multifocal leukoencephalopathy (PML) and cystitis, respectively. We have established a specific and sensitive method to identify SV40 sequence in DNA extracted from histological sections, using PCR followed by Southern hybridization to probes specific to the large T region. We found SV40 large T antigen sequences in all brain tumor types investigated. High frequencies were found in low-grade astrocytomas, anaplastic astrocytomas and secondary glioblastomas derived thereof (13/22, 59%) while somewhat lower frequencies were found in gemistocytic astrocytomas (9/28, 32%) and oligodendrogliomas (3/12, 25%). Primary glioblastomas, giant cell glioblastomas, and gliosarcomas, which clinically develop de novo, contained SV40 sequences in 11-25% of cases. Presence of viral DNA was also observed in pediatric brain tumors, including ependymomas (9/16, 56%), choroid plexus papillomas (6/16, 38%), and medulloblastomas (5/17, 29%). In 8 tumor biopsies with SV40 sequences, the adjacent normal brain tissue was also analyzed but was devoid of viral DNA in all but one case. BK and JC virus sequences were rarely detected, the overall frequencies being 3% and 2%, respectively. It remains to be shown whether the presence of SV40 contributes significantly to malignant transformation or whether certain human neoplasms provide a microenvironment that favors viral replication in humans with latent SV40 infection.

Adolescent↗
PubMed2003

Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3'-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas.

Glioblastomas frequently carry mutations in the PTEN tumor suppressor gene on 10q23.3. The tumor suppressor properties of Pten are closely related to its inhibitory effect on the phosphatidyl-inositol-3'-kinase (Pi3k)-dependent activation of protein kinase B (Akt) signalling. Here, we report on the analysis of 17 genes related to the Pi3k/Akt signalling pathway for genetic alteration and aberrant expression in a series of 103 glioblastomas. Mutation, homozygous deletion or loss of expression of PTEN was detected in 32% of the tumors. In contrast, we did not find any aberrations in the inositol polyphosphate phosphatase like-1 gene (INPPL1), whose gene product may also counteract Pi3k-dependent Akt activation. Analysis of genes encoding proteins that may activate the pathway upstream of Pi3k revealed variable fractions of tumors with EGFR amplification (31%), PDGFRA amplification (8%), and IRS2 amplification (2%). The protein tyrosine kinase 2 (PTK2/FAK1) gene was neither amplified nor overexpressed at the mRNA level. Investigation of three genes encoding catalytic subunits of Pi3k (PIK3CA, PIK3CD, and PIK3C2B) revealed amplification of PIK3C2B (1q32) in 6 tumors (6%). Overexpression of PIK3C2B mRNA was detected in 4 of these cases. PIK3CD (1p36.2) and PIK3CA (3q26.3) were not amplified but PIK3CD mRNA was overexpressed in 6 tumors (6%). Amplification and overexpression of AKT1 was detected in a single case of gliosarcoma. The IRS1, PIK3R1, PIK3R2, AKT2, AKT3, FRAP1, and RPS6KB1 genes were neither amplified nor overexpressed in any of the tumors. Taken together, our data indicate that different genes related to the Pi3k/Akt signalling pathway may be aberrant in glioblastomas.

Adolescent↗
PubMed2006

Characterization of glioblastomas in young adults.

Most adult glioblastoma multiformes (GBMs) present in patients 45-70 years old; tumors occurring at the extremes of the adult age spectrum are uncommon, and seldom studied. We hypothesized that young-adult GBMs would differ from elderly-adult and from pediatric GBMs. Cases were identified from years 1997 to 2005. Demographic and histological features, MIB-1 and TP53 immunohistochemical findings and epidermal growth factor receptor (EGFR) amplification status by fluorescence in situ hybridization were compiled and correlated with survival. Twenty-eight (74%) of our 38 young-adult GBM patients had primary de novo tumors, two of which occurred in patients with cancer syndromes. Two additional GBMs were radiation-induced and eight (21%) were secondary GBMs. Seven patients were identified as long-term (>3 years) survivors. Six of 38 cases manifested unusual morphological features, including three epithelioid GBMs, one rhabdoid GBM, one gliosarcoma and one small cell GBM containing abundant, refractile, eosinophilic inclusions. MIB-1 index emerged as the most important prognosticator of survival (P < 0.005). Although there was a trend between extent of necrosis, TP53 immunohistochemical expression, and EGFR amplification status and survival, none reached statistical significance. GBMs in young adults are a more inhomogeneous tumor group than GBMs occurring in older adult patients and show features that overlap with both pediatric and adult GBMs.

Adolescent↗
PubMed2005

Enantioselective metabolism and cytotoxicity of R-ifosfamide and S-ifosfamide by tumor cell-expressed cytochromes P450.

The anticancer prodrug ifosfamide (IFA) contains a chiral phosphorous atom and is administered in the clinic as a racemic mixture of R-IFA and S-IFA. Hepatic cytochrome P450 (P450) enzymes exhibit enantioselective preferences in the metabolism of R-IFA and S-IFA; however, the impact of this selectivity on P450-dependent anticancer activity is not known. Presently, the metabolism and cytotoxicity of R-IFA and S-IFA were determined in 9L gliosarcoma and Chinese hamster ovary tumor cells expressing an IFA-activating P450 enzyme and by in vitro steady-state kinetic analysis using cDNA-expressed P450 enzymes. Tumor cells expressing P450 enzyme CYP3A4 were the most sensitive to R-IFA cytotoxicity, whereas tumor cells expressing CYP2B1 or CYP2B6 were most sensitive to cyclophosphamide (CPA), an isomer of IFA. Correspondingly, CYP3A4-expressing cells and cDNA-expressed CYP3A4 metabolized R-IFA to yield the active, 4-hydroxylated metabolite at a 2- to 3-fold higher rate than they metabolized S-IFA or CPA. CYP2B cells and cDNA-expressed CYP2B enzymes metabolized CPA almost exclusively by 4-hydroxylation, whereas R-IFA and S-IFA were substantially converted to inactive, N-dechloroethylated metabolites. Further investigation revealed that CYP3A1, a rat enzyme, exhibited superior kinetic properties compared with the human enzyme CYP3A4, with R-IFA and S-IFA both metabolized with high catalytic efficiency by 4-hydroxylation and with a K(m) value of 200 microM, approximately 5-fold lower than CYP3A4. Based on these kinetic parameters and metabolic profiles, R-IFA is expected to exert greater anticancer activity than S-IFA or CPA against tumors that express CYP3A enzymes, whereas tumors expressing CYP2B enzymes may be more sensitive to CPA treatment.

Acetaldehyde↗
PubMed2001

Cyclophosphamide induces caspase 9-dependent apoptosis in 9L tumor cells.

Cyclophosphamide (CPA), a widely used oxazaphosphorine anti-cancer prodrug, is inactive until it is metabolized by cytochrome P450 to yield phosphoramide mustard and acrolein, which alkylate DNA and proteins, respectively. Tumor cells transduced with the human cytochrome P450 gene CYP2B6 are greatly sensitized to CPA, however, the pathway of CPA-induced cell death is unknown. The present study investigates the cytotoxic events induced by CPA in 9L gliosarcoma cells retrovirally transduced with CYP2B6, or induced in wild-type 9L cells treated with mafosfamide (MFA) or 4-hydroperoxyifosfamide (4OOH-IFA), chemically activated forms of CPA and its isomer ifosfamide. CPA and MFA were both shown to effect tumor cell death by stimulating apoptosis, as evidenced by the induction of plasma membrane blebbing, DNA fragmentation, and cleavage of the caspase 3 and caspase 7 substrate poly(ADP-ribose) polymerase (PARP) in drug-treated cells. Caspase 9 was identified as the regulatory upstream caspase activated in 9L cells treated with CPA, MFA, or 4OOH-IFA, implicating the mitochondrial apoptotic pathway in oxazaphosphorine-induced tumor cell death. Correspondingly, expression of the mitochondrial proapoptotic factor Bax enhanced caspase 9 activation, plasma membrane blebbing, and drug-induced cytotoxicity. Conversely, overexpression of the mitochondrial antiapoptotic factor Bcl-2 blocked caspase 9 activation, leading to an inhibition of drug-induced plasma membrane permeability and blebbing, terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity, PARP cleavage, Annexin V positivity, and drug-induced cell death. Although Bcl-2 thus blocked the cytotoxic effects of activated CPA, it did not inhibit the drug's cytostatic effects. CPA induced S-phase cell cycle arrest followed by conversion to an apoptotic pre-G1 state in wild-type 9L cells; by contrast, Bcl-2-expressing 9L cells accumulated in G2/M in response to CPA treatment. Intratumoral expression of Bcl-2 and related family members, including both apoptotic and antiapoptotic factors, is thus an important determinant of the responsiveness of tumor cells to CPA and ifosfamide, both in the context of conventional chemotherapy and in patients sensitized to these oxazaphosphorine drugs by the use of cytochrome P450-based gene therapy.

Animals↗