Increased age of transformed mouse neural progenitor/stem cells recapitulates age‐dependent clinical features of human glioma malignancy

Increasing age is the most robust predictor of greater malignancy and treatment resistance in human gliomas. However, the adverse association of clinical course with aging is rarely considered in animal glioma models, impeding delineation of the relative importance of organismal versus progenitor cell aging in the genesis of glioma malignancy. To address this limitation, we implanted transformed neural stem/progenitor cells (NSPCs), the presumed cells of glioma origin, from 3‐ and 18‐month‐old mice into 3‐ and 20‐month host animals. Transplantation with progenitors from older animals resulted in significantly shorter (P ≤ 0.0001) median survival in both 3‐month (37.5 vs. 83 days) and 20‐month (38 vs. 67 days) hosts, indicating that age‐dependent changes intrinsic to NSPCs rather than host animal age accounted for greater malignancy. Subsequent analyses revealed that increased invasiveness, genomic instability, resistance to therapeutic agents, and tolerance to hypoxic stress accompanied aging in transformed NSPCs. Greater tolerance to hypoxia in older progenitor cells, as evidenced by elevated HIF‐1 promoter reporter activity and hypoxia response gene (HRG) expression, mirrors the upregulation of HRGs in cohorts of older vs. younger glioma patients revealed by analysis of gene expression databases, suggesting that differential response to hypoxic stress may underlie age‐dependent differences in invasion, genomic instability, and treatment resistance. Our study provides strong evidence that progenitor cell aging is responsible for promoting the hallmarks of age‐dependent glioma malignancy and that consideration of progenitor aging will facilitate development of physiologically and clinically relevant animal models of human gliomas.     

Multimodal imaging of neural progenitor cell fate in rodents

For clinical application of stem cell-based therapies, noninvasive detection of applied stem cells is of high importance. We report on the feasibility of detecting implanted neural progenitor cells (NPCs) noninvasively and follow their fate and functional status by sequential multimodal molecular imaging and reporter gene technology. We investigated C17.2 cells stably expressing herpes simplex virus type 1-thymidine kinase (HSV-1-tk) and green fluorescent protein (gfp) (C17.2-tkIRESgfp = C17.2-TIG) or HSV-1-tk, gfp, and firefly luciferase (luc) (C17.2-lucIREStkgfp = C17.2-LITG) and determined the detection sensitivity of positron emission tomography (PET) and bioluminescence imaging (BLI) for these cells in culture and in vivo in subcutaneous and intracranial glioma models. In addition, PET and BLI were used to further investigate and follow the fate of implanted C17.2-LITG cells in an intracranial glioma model. We show that both imaging modalities are sensitive in detecting reporter gene expressing NPCs; however, PET, by the use of 9-[4-[(18)F]fluoro-3-hydroxymethyl)butyl]guanine ([(18)F]FHBG), detects NPCs only at sites of disrupted blood-brain barrier. Furthermore, both imaging modalities can be used to detect stem cell fate and migration and indicate excessive proliferation and aberrant migration. In conclusion, multimodal imaging can be used for longitudinal noninvasive monitoring of grafted NPCs in rodents.     

The impact of age on oncogenic potential: tumor‐initiating cells and the brain microenvironment

Paradoxically, aging leads to both decreased regenerative capacity in the brain and an increased risk of tumorigenesis, particularly the most common adult‐onset brain tumor, glioma. A shared factor contributing to both phenomena is thought to be age‐related alterations in neural progenitor cells (NPCs), which function normally to produce new neurons and glia, but are also considered likely cells of origin for malignant glioma. Upon oncogenic transformation, cells acquire characteristics known as the hallmarks of cancer, including unlimited replication, altered responses to growth and anti‐growth factors, increased capacity for angiogenesis, potential for invasion, genetic instability, apoptotic evasion, escape from immune surveillance, and an adaptive metabolic phenotype. The precise molecular pathogenesis and temporal acquisition of these malignant characteristics is largely a mystery. Recent studies characterizing NPCs during normal aging, however, have begun to elucidate mechanisms underlying the age‐associated increase in their malignant potential. Aging cells are dependent upon multiple compensatory pathways to maintain cell cycle control, normal niche interactions, genetic stability, programmed cell death, and oxidative metabolism. A few multi‐functional proteins act as ‘critical nodes’ in the coordination of these various cellular activities, although both intracellular signaling and elements within the brain environment are critical to maintaining a balance between senescence and tumorigenesis. Here, we provide an overview of recent progress in our understanding of how mechanisms underlying cellular aging inform on glioma pathogenesis and malignancy.     

Neural stem cell-derived factors inhibit the growth and invasion of U87 stem-like cells in vitro

Glioma is one of the most common and aggressive tumors in the brain. Significant attention has been paid to the potential use of neural stem/progenitor cells (NSCs/NPCs) as delivery vehicles to cure gliomas. However, whether the NSCs/NPCs or the factors they produced could make a contribution still remains to be seen. In this study, we focused on the inhibitory effects of the factors produced by NSCs/NPCs on the biological behavior of the glioma stem-like cell in vitro. The human glioma cell line U87 was selected and the U87 stem-like cells were addressed. After being cultured in the NSC condition medium (NSC-CM), the viability and proliferation of U87 stem-like cells were significantly reduced. The invasion of U87 stem-like cells and the migration of U87 cells were also significantly decreased. However, no significant change was observed in regard to the astrocytic differentiation of U87 stem-like cells. These indicated that NSCs/NPCs produced some factors and had an inhibitory effect on the growth and invasion but not the terminal differentiation of U87 stem-like cells. It is worth paying attention to NSCs/NPCs as a high-potential candidate for glioma treatment.     

Antitumour and pro-apoptotic actions of highly unsaturated fatty acids in glioma

The highly unsaturated fatty acids (HUFA) of the n-6 and n-3 series are involved in cell signalling in normal and transformed cells and have recently been associated with pathways leading to tumour cell death. The antitumour activity of three HUFA (arachidonic acid, gamma linolenic acid and eicosapentaenoic acid) were studied in glioma cells and tissue. Using five glioma models, including primary cell suspensions prepared from 46 human glioma samples and an in vivo rat C6 glioma model, we obtained evidence that, following exposure to HUFA, either administered into the medium surrounding human glioma cells or in 16 preparations of multicellular spheroids derived from human and rodent glioma cell lines (C6, MOG, U87, U373) or administered intra-tumourally by infusion using osmotic mini-pumps in 48 rats, glioma regression and apoptosis were detected. Additionally, synergy between gamma irradiation and HUFA administration was observed in 13 experiments analyzing C6 glioma cell apoptosis in vitro. These pro-apoptotic and antiproliferative activities were observed using both C18 and C20 fatty acids of the n-6 and n-3 series, but not when saturated and monounsaturated C18 and C20 fatty acid preparations were used. In the glioma infusion model, in addition to the apoptosis detected in glioma tissue infused with HUFA for 3-7 days, preservation of normal neural tissue and vasculature in adjacent brain was observed. Also, there was little evidence of acute inflammatory infiltration in regressing tumours. Our findings suggest that intraparenchymal infusion of HUFA may be effective in stimulating glioma regression.     

Transformation of mammalian cells by alpha particles.

Mammalian cells in culture have been shown here for the first time to be transformed by alpha irradiation. Mouse embryo (C3H 10T1/2) cells were transformed with 5.6 MeV alpha particles from a Tandem Van de Graaff machine. Malignant tumours were induced following inoculation of the transformed cells into syngeneic hosts. Unirradiated control cells failed to produce tumours. The morphology of the transformed foci was similar to that obtained by X-rays and chemicals but different from virally transformed cells. The transformation frequency increased approximately as the cube of the dose to a maximum of about 4 per cent ofthe surviving cells which occurred between 1.5 and 2.5 x 10(7) alpha particles per cm2 (205-342 rad). It appears that alpha particle irradiation may exert a direct effect on the genome of the cell to produce malignancy without any external immunological or hormonal influences.     

Production of a low molecular weight growth inhibitory factor by adenovirus 12-transformed cells.

Malignant rodent cells transformed by human adenovirus 12 produce a potent cell growth inhibitory factor. The cell growth inhibitory factor inhibits the growth of and DNA synthesis in normal fibroblasts in vitro. Extent of the production of the cell growth inhibitory factor appears to be proportional to that of the malignancy of the transformed cells. C57AT1-AB cells, an adenovirus 12-transformant of C57BL/6 mouse origin, are highly tumorigenic in the syngeneic and allogeneic mice. The cell growth inhibitory factor produced by these cells was characterized for the physicochemical properties; the cell growth-inhibitory activity was quantitatively recovered in the filtrates of YM-2 membrane (M(r) less than 1,000), resistant to the heat treatments at 56 degrees C for 30 min and 100 degrees C for 5 min, and extractable by ethyl acetate under acid-condition. These results suggest that the cell growth inhibitory factor may be lipid or oligopeptides.     

Generation of neural progenitor cells by chemical cocktails and hypoxia

Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, Repsox, an inhibitor of TGF-β pathways), under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs regarding their proliferative and self-renewing abilities, gene expression profiles, and multipotency for different neuroectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase, and TGF-β pathways show similar efficacies for ciNPC induction. Moreover, ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemical cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors.     

Isolation and characterization of in vitro and in vivo functions of a tumor-specific T suppressor cell clone from a BALB/c mouse bearing the syngeneic ADJ-PC-5 plasmacytoma

A spontaneously transformed T suppressor (Ts) clone, A12-D11/t, is described which arose from the antigen-specific Ts clone A12-D11 isolated from cells of the inguinal lymph node of a BALB/c mouse bearing the syngeneic plasmacytoma ADJ-PC-5. A12-D11/t Ts cells suppress in vitro specifically a primary syngeneic cytotoxic antitumor response with the consequence that no ADJ-PC-5-specific cytotoxic T cells can be generated. The in vivo effects of A12-D11/t Ts cells were studied by injecting them into BALB/c mice. Spleen cells from those mice subsequently failed to respond against ADJ-PC-5 plasmacytoma cells, whereas their response against other syngeneic BALB/c tumors remained unaffected. By using a model system which allows us to study the host's immune reactions to the antigenic load corresponding to initial stages of tumorigenesis, it has been shown previously that ADJ-PC-5-specific Ts cells are activated before the antigen threshold for the activation of cytotoxic T cells is reached. Regarding its phenotype and specificity, the A12-D11/t Ts clone seems to be the exact counterpart of such a Ts cell, and is therefore of special interest in the study of the role of Ts cells preventing immunity against a growing tumor.     

Radiation Sensitivity of GL261 Murine Glioma Model and Enhanced Radiation Response by Flavopiridol

Response of a solid tumor to radiation treatment depends, in part, on the intrinsic radiosensitivity of tumor cells, the proliferation rate of tumor cells between radiation treatments and the hypoxic state of the tumor cells. A successful radiosensitizing agent would target S-phase cells and hypoxia. Recently, we demonstrated the anti-tumor effects of flavopiridol in the GL261 murine glioma model might involve 1) recruitment of tumor cells to S-phase (Newcomb et al., Cell Cycle 2004; 3:230-234) and 2) an anti-angiogenic effect on the tumor vasculature by downregulation of hypoxia-inducible factor -1? (HIF-1?) (Newcomb et al., Neuro-Oncology 2005; 7:225-235). Given that flavopiridol has demonstrated radiosensitizing activity in several murine tumor models, we tested whether it would enhance the response of GL261 tumors to radiation. In the present study, we evaluated the intrinsic radiation sensitivity of the GL261 glioma model using the tumor control/cure dose of radiation assay (TCD50). We found that a single dose of 65 Gy (CI 57.1-73.1) was required to cure 50% of the tumors locally. Using the tumor growth delay assay, fractionated radiation (5 fractions of 5 Gy over 10 days) combined with flavopiridol (5 mg/kg) given three times weekly for 3 cycles produced a significant growth delay. Our results indicate that the GL261 murine glioma model mimics the radioresistance encountered in human gliomas, and thus should prove useful in identifying promising new investigational radiosensitizers for use in the treatment of glioma patients.     

Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

Background

Neural progenitor cells (NPCs) in the developing neuroepithelium are regulated by intrinsic and extrinsic factors. There is evidence that NPCs form a self-supporting niche for cell maintenance and proliferation. However, molecular interactions and cell-cell contacts and the microenvironment within the neuroepithelium are largely unknown. We hypothesized that cellular proteases especially those associated with the cell surface of NPCs play a role in regulation of progenitor cells in the brain.

Methodology/Principal Findings

In this work, we show that NPCs, isolated from striatal anlage of developing rat brain, express hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2) that are cell surface-linked serine protease inhibitors. In addition, radial glia cells derived from mouse embryonic stem cells also express HAI-1 and HAI-2. To study the functional significance of HAI-1 and HAI-2 in progenitor cells, we modulated their levels using expression plasmids or silencing RNA (siRNA) transfected into the NPCs. Data showed that overexpression of HAI-1 or HAI-2 decreased cell proliferation of cultured NPCs, whilst their siRNAs had opposite effects. HAI-1 also influenced NPC differentiation by increasing the number of glial fibrillary acidic protein (GFAP) expressing cells in the culture. Expression of HAI-1 in vivo decreased cell proliferation in developing neuroepithelium in E15 old animals and promoted astrocyte cell differentiation in neonatal animals. Studying the regulation of HAI-1, we observed that Bone morphogenetic protein-2 (BMP-2) and BMP-4 increased HAI-1 levels in the NPCs. Experiments using HAI-1-siRNA showed that these BMPs act on the NPCs partly in a HAI-1-dependent manner.

Conclusions

This study shows that the cell-surface serine protease inhibitors, HAI-1 and HAI-2 influence proliferation and cell fate of NPCs and their expression levels are linked to BMP signaling. Modulation of the levels and actions of HAI-1 in NPCs may be of a potential value in stem cell therapies in various brain diseases.     

Proteolytic processing of SDF-1 α by matrix metalloproteinase-2 impairs CXCR4 signaling and reduces neural progenitor cell migration

Neural stem cells and neural progenitor cells (NPCs) exist throughout life and are mobilized to replace neurons, astrocytes and oligodendrocytes after injury. Stromal cell-derived factor 1 (SDF-1, now named CXCL12) and its receptor CXCR4, an α-chemokine receptor, are critical for NPC migration into damaged areas of the brain. Our previous studies demonstrated that immune activated and/or HIV-1-infected human monocyte-derived- macrophages (MDMs) induced a substantial increase of SDF-1 production by human astrocytes. However, matrix metalloproteinase (MMP)-2, a protein up-regulated in HIV-1-infected macrophages, is able to cleave four amino acids from the N-terminus of SDF-1, resulting in a truncated SDF-1(5-67). In this study, we investigate the diverse signaling and function induced by SDF-1 α and SDF-1(5-67) in human cortical NPCs. SDF-1(5-67) was generated by incubating human recombinant SDF-1α with MMP-2 followed by protein determination via mass spectrometry, Western blotting and ELISA. SDF-1α induced time-dependent phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, Akt-1, and diminished cyclic adenosine monophosphate (cAMP). In contrast, SDF-1(5-67) failed to induce these signaling. SDF-1α activation of CXCR4 induced migration of NPCs, an effect that is dependent on ERK1/2 and Akt-1 pathways; whereas SDF-1(5-67) failed to induce NPC migration. This observation provides evidence that MMP-2 may affect NPC migration through post-translational processing of SDF-1α.     

Neural precursor cells differentiated from mouse embryonic stem cells relieve symptomatic motor behavior in a rat model of Parkinson's disease

Pluripotent embryonic stem (ES) cells are the most versatile cells, with the potential to differentiate into all types of cell lineages including neural precursor cells (NPCs), which can be expanded in large numbers for significant periods of time to provide a reliable cell source for transplantation in neurodegenerative disorders such as Parkinson's disease (PD). In the present study, we used the MESPU35 mouse ES cell line, which expresses enhanced green fluorescent protein that enables one to distinguish between transplanted cells and cells of host origin. Embryoid bodies (EBs) were formed and were induced to NPCs in N2 selection medium plus fibronectin. Praxiology and immunohistochemistry methods were used to observe the survival, differentiation, and therapeutic effect of NPCs after grafted into the striatum of PD rats. We found that mouse ESc were differentiated into nestin-positive NPCs 6 days after the EBs formed and cultured in the N2 selection medium. The number of survival NPCs was increased significantly by fibronectin. About 23.76+/-2.29% of remaining cells were tyrosine hydroxylase (TH)-positive 12 days after NPCs were cultured in N2 selective medium. The survival rates of NPCs were 2.10+/-0.41% and about 90.90+/-3.00% of the engrafted NPCs were TH-positive 6 weeks after transplantation into the striatum of PD rats. The rotation of PD rats was relieved 3 weeks after the NPCs transplantation and this effect was kept for at least 6 weeks. It suggests that most of the survival NPCs derived from ES cells differentiated into TH-positive neurons after grafted into the striatum of PD rats, which produces therapeutic effect on PD.     

CD8+ T-cell mediated anti-tumor responses cross-reacting against 9L and RT2 rat glioma cell lines

We have shown that vaccination of animals with two distinct commonly used glioma cell lines, 9L and RT2, generated cross-reactive cellular anti-tumor immunity. Peripheral vaccination with either cell line 9L or RT2 resulted in MHC Class I restricted effector cells capable of in vitro cytolytic activity against both target 9L and RT2 cells but not the syngeneic F98 glioma cell line. In vitro cross-reactive cytolytic activity could be measured for as long as 6 months from the time of initial vaccination. Fractionation of splenic effector cells revealed the cytolytic activity to be CD8+ T-cell mediated but required CD4+ T-cells for effective antigen presentation. Anti-tumor immunity generated after vaccination with either 9L or RT2 was completely protective against subsequent subcutaneous inoculation of animals with either 9L or RT2 cells and resulted in prolonged survival in animals inoculated intracranially with either cell line. Our results suggest that despite the different methods used in their derivation, 9L and RT2 glioma cells share a common glioma antigen recognized by the cellular arm of the immune response.     

Relationship between Initial Telomere Length,Initial Telomerase Activity,Age, and Replicative Capacity of Nucleus Pulposus Chondrocytes in Human Intervertebral Discs: What Is a Predictor of Replicative Potential?

There is evidence that telomere length (TL), telomerase activity (TA), and age are related to the replicative potential of human nucleus pulposus chondrocytes (NPCs). However, it has not yet been established if any of these factors can serve as predictors of the replicative potential of NPCs. To establish predictors of the replicative potential of NPCs, we evaluated potential relationships between replicative capacity of NPCs, initial TL (telomere length at the first passage), initial TA (telomerase activity at the first passage), and age. Nucleus pulposus specimens were obtained from 14 patients of various ages undergoing discectomy. NPCs were serially cultivated until the end of their replicative lifespans. Relationships among cumulative population doubling level (PDL), initial TL, initial TA, and age were analyzed. Initial TA was negatively correlated with age (r = -0.674, P = 0.008). However, no correlation between initial TL and age was observed. Cumulative PDL was also negatively correlated with age (r = -0.585, P = 0.028). Although the cumulative PDL appeared to increase with initial TL or initial TA, this trend was not statistically significant. In conclusion, age is the sole predictor of the replicative potential of human NPCs, and replicative potential decreases with age. Initial TL and initial TA are not predictors of replicative potential, and can serve only as reference values.     

Specificities of killing by cytotoxic lymphocytes generated in vivo and in vitro to syngeneic SV40 transformed cells.

Cell-mediated immunity to SV40-transformed C3H and C3H-SW cell lines was measured by using both 51Cr and 125IUdR release assays. Killing by cytotoxic cells generated on in vitro sensitization of immune spleen cells with syngeneic SV40 cells by either assay is specific for syngeneic SV40 transformants. Cytolysis mediated by in vitro sensitized cells is ablated by treatment of the effector cells with anti-theta serum and complement. Intraperitoneal immunization with syngeneic SV40 cells yields two distinct killer-cell populations in the peritoneal exudate when assayed by 125IUdR release. The first, nylon wool nonadherent and sensitive to anti-theta and complement, is indistinguishable from the killers generated in vitro. The second population, present in larger numbers and more efficient on a per-cell basis in killing of SV40 targets than the first, is nylon adherent and is not removed by treatment with anti-theta and complement. This second population will kill any SV40 transformed target, whether syngeneic or allogeneic. The possible roles of T cell and non-T cell effectors in rejection of syngeneic SV40 tumors are discussed.     

Human epidermal keratinocytes retain radiation resistance following in vitro immortalization and malignant transformation

The radiobiology of human tumors suggests that multiple factors are involved in clinical radioresponsiveness. To date, no direct experimental evidence is available to correlate intrinsic cellular radiosensitivity with the steps of malignant transformation. We developed an in vitro multistage model of epithelial neoplasia using human epidermal keratinocytes to examine the effects of malignant transformation on radiation response. These cells were first immortalized as a result of infection with a hybrid virus (adenovirus 12 and simian virus 40) and subsequently transformed either by infection with a second virus (Kirsten murine sarcoma virus) or by treatment with a chemical carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide). We demonstrate that primary human epidermal keratinocytes were radiation resistant (D0 = 2.24 Gy) as compared with human fibroblasts (D0 = 1.45 Gy) and that this resistance was retained in the immortalized as well as the transformed cell lines. These findings present direct experimental evidence that radiation sensitivity of malignant human keratinocytes is an intrinsic property of the precursor cell that may be conserved through the stages of neoplastic transformation.