Dennis F. Deen

The 9L rat brain tumor: Description and application of an animal model

SummaryAnimal models allow determination of tumor response to anticancer agents under various experimental conditions. The chemically induced 9L rat brain tumor has been developed as both in vivo and in vitro models. Animal survival, clonogenic cell survival, and tumor growth delay provide means to measure the effectiveness of treatment modalities in this tumor model. Monolayer cultures, multicellular spheroid cultures, brain tumors, and flank tumors have been used to study the influence of different biological entities of the 9L model on the response to treatment with radiation and/or BCNU (1,3- bis (2-chloroethyl)-1-nitrosourea).ZusammenfassungDie Behandlung von Tumoren unterliegt den Einflüssen verschiedener Umgebungsfaktoren, die an Tiertumormodellen erforscht werden konnten. Für den chemisch induzierten 9L Hirntumor der Ratte wurden in vivo- and in vitro-Systeme entwickelt. Überlebensraten von Tieren oder clonogenen Zellen und Wachstumsverzögerung von Tumoren können gemessen werden, um die Wirksamkeit verschiedener Tumorbehandlungen zu bestimmen. Monolayer- und Spheroidkulturen sowie intracerebral oder subcutan wachsende Tumoren wurden verwendet, um den Einfluß unterschiedlicher Tumorgestalt auf die Wirkung von Strahlen und/oder BCNU (1,3- bis (2-chloroethyl)-1-nitrosourea) bei 9L Zellen zu demonstrieren.

Distribution in brain of liposomes after convection enhanced delivery; modulation by particle charge, particle diameter, and presence of steric coating

We have investigated the role of diameter, charge, and steric shielding on the brain distribution of liposomes infused by convection enhanced delivery (CED) using both radiolabeled and fluorescent-labeled particles. Liposomes of 40 and 80-nm diameter traveled the same distance but penetrated significantly less than a 10-kDa dextran; whereas 200-nm-diameter liposomes penetrated less than 80 nm liposomes. A neutral liposome shielded by polyethylene glycol (PEG; 2 kDa; 10% by mole) penetrated significantly farther than an unshielded liposome. Even when shielded with PEG, positive surface charge (10% by mole) significantly reduced the penetration radius compared to a neutral or negative charged liposome (10% by mole). A mathematical CED model including a term for liposome cell binding was applied to analyze the radius of particle penetration. Neutral liposomes had a binding constant of k=0.0010+/-0.0002 min-1, whereas for positive charged liposomes k increased 50-fold. The binding constant was independently verified using a degradable lipid radiolabel that eliminated from the brain with a 9.9+/-2.0 h half-life, equivalent to the calculated elimination constant k=0.0012+/-0.0002 min-1. During CED, liposomes accumulated in a subpopulation of perivascular cells within the brain. A non-degradable lipid radiolabel showed that lipid components remained within these perivascular brain cells for at least 2 days. To reduce this uptake, 100-fold molar excess of non-labeled liposomes were co-infused with labeled liposomes, which significantly increased liposome penetration. These studies suggest that optimization of therapeutic CED using particles such as drug-loaded liposomes, polymeric nanoparticles, non-viral DNA complexes, and viruses will require a strategy to overcome particle binding and clearance by cells within the CNS.

p53-Dependent G1 arrest and p53-independent apoptosis influence the radiobiologic response of glioblastoma

PURPOSE Loss of the p53 tumor suppressor gene has been associated with tumor progression, disease relapse, poor response to antineoplastic therapy, and poor prognosis in many malignancies. We have investigated the contribution of p53-mediated radiation-induced apoptosis and G1 arrest to the well described radiation resistance of glioblastoma multiforme (GM) cells. METHODS AND MATERIALS Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Isogenic derivatives of glioblastoma cells differing only in their p53 status were generated using a retroviral vector expressing a dominant negative mutant of p53. Radiation-induced apoptosis was assayed by Fluorescence-activated cell sorter (FACS) analysis, terminal deoxynucleotide transferase labeling technique, and chromatin morphology. Cells were synchronized in early G1 phase and mitotic and labeling indices were measured. RESULTS Radiation-induced apoptosis of GM cells was independent of functional wild-type p53 (wt p53). Decreased susceptibility to radiation-induced apoptosis was associated with lower alpha values characterizing the shoulder of the clonogenic radiation survival curve. Using isogenic GM cells differing only in their p53 activity, we found that a p53-mediated function, radiation-induced G1 arrest, could also influence the value of alpha and clonogenic radiation resistance. Inactivation of wt p53 function by a dominant negative mutant of p53 resulted in a significantly diminished alpha value with no alteration in cellular susceptibility to radiation-induced apoptosis. The clonal derivative U87-LUX.8 expressing a functional wt p53 had an alpha (Gy-1) value of 0.609, whereas the isogenic clonal derivative U87-175.4 lacking wt p53 function had an alpha (Gy-1) value of 0.175. CONCLUSION We conclude that two distinct cellular responses to radiation, p53-independent apoptosis and p53-dependent G1-arrest, influence radiobiological parameters that characterize the radiation response of glioblastoma cells. Further understanding of the molecular basis of GM radiation resistance will lead to improvement in existing therapeutic modalities and to the development of novel treatment approaches.

Barriers to carrier mediated drug and gene delivery to brain tumors.

Brain tumor patients face a poor prognosis despite significant advances in tumor imaging, neurosurgery and radiation therapy. Potent chemotherapeutic drugs fail when used to treat brain tumors because biochemical and physiological barriers limit drug delivery into the brain. In the past decade a number of strategies have been introduced to increase drug delivery into the brain parenchyma. In particular, direct drug administration into the brain tumor has shown promising results in both animal models and clinical trials. This technique is well suited for the delivery of liposome and polymer drug carriers, which have the potential to provide a sustained level of drug and to reach cellular targets with improved specificity. We will discuss the current approaches that have been used to increase drug delivery into the brain parenchyma in the context of fluid and solute transport into, through and from the brain, with a focus on liposome and polymer drug carriers.

BCNU and x-ray therapy of intracerebral 9L rat tumors

Abstract Combination therapy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and X-rays was applied to the 9L rat brain tumor model. BCNU alone (13.3 mg/kg) and X-rays alone (2,000 rad/whole brain) were given on day 16 postimplant. The two therapies were combined in three schedules with BCNU given 6 hours before, 6 hours after, and immediately preceding X-ray treatment. While either modality alone significantly increased survival, the combined therapies produced increased life spans (ILS) of 235 to 430%. The combined therapies also resulted in cure rates as high as 60%. Some toxicity was encountered as evidenced by interstitial pulmonary fibrosis when “cured” and treated non-tumor bearing animals were autopsied on day 122 or day 125.

P53 FUNCTION INFLUENCES THE EFFECT OF FRACTIONATED RADIOTHERAPY ON GLIOBLASTOMA TUMORS

PURPOSE Glioblastoma multiforme brain tumors (GM) are treated with a spectrum of fractionation regimens based on the clinical and anatomical characteristics of the tumor but rarely based on the molecular characteristics of the individual neoplasm. This study tests the hypothesis that the response of cell lines derived from GM to fractionated radiotherapy depends on the function of wild-type p53 (wt p53), a tumor suppressor gene frequently mutated in GM tumors. METHODS & MATERIALS Isogenic derivatives of glioblastoma cells differing only in p53 function were prepared using a retroviral vector expressing a dominant negative mutant of p53 (mt p53). Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Apoptosis was assayed by a terminal deoxynucleotide transferase-labeling technique and chromatin morphology. RESULTS We have previously reported the generation of isogenic GM cell lines differing only in p53 function. U87-175.4, lacking wt p53 function, had a significantly lower alpha/beta value than U87-LUX.8, expressing functional wt p53, leading us to hypothesize that fractionated irradiation would preferentially spare GM cells harboring mt p53 compared with those expressing functional, wt p53. Survival curves following either 2.0 Gy or 3.5 Gy/fraction demonstrated that lack of functional wt p53 was associated with resistance to fractionated irradiation. Radiation-induced apoptosis could not account for the observed differences in clonogenic survival. Rather, our data suggested that a deficit in the G1-checkpoint contributed to increased resistance to fractionated irradiation of cells expressing mutant p53. CONCLUSIONS The effect of fractionated radiotherapy in GM may depend on the function of the tumor suppressor gene p53. A potential clinical consequence of these findings is that hyperfractionation regimens may provide a therapeutic advantage specifically for tumors expressing wt p53 whereas a radiotherapy course of fewer, larger fractions may be appropriate for the treatment of tumors carrying p53 mutations. Further studies are needed to confirm our proposal that the p53 status of GM tumors can be used to guide our choice of fractionation schemes.

Cytotoxicity and cell-cycle effects of paclitaxel when used as a single agent and in combination with ionizing radiation☆

PURPOSE This study aimed to determine the extent of paclitaxel-induced cytotoxicity and cell-cycle perturbations when used alone and in combination with radiation in human glioma cells. METHODS AND MATERIALS The effect of paclitaxel alone on three human glioma cells lines--SF-126, U-87 MG, and U-251 MG--was assessed after 24, 48, 72, or 96 h treatment. For experiments in combination with radiation, cells were exposed to either a long (48-h) or short (8-h) duration of paclitaxel treatment prior to irradiation. Cell survival was determined by clonogenic assay. Cell cycle perturbations were assessed by using flow cytometry to measure the proportion of cells in G1, S, and G2/M phases. RESULTS When cells were treated with paclitaxel alone for > or = 24 h, cytotoxicity increased up to a threshold dose, after which it plateaued. When treatment duration was < or = 24 h, cytotoxicity was appreciably greater in U-251 MG cells than in SF-126 and U-87 MG cells. After 24 h of paclitaxel treatment, cells in plateau phase growth had increased survival compared to cells in log phase growth. In contrast, after 8 h paclitaxel treatment, mitotic cells had reduced survival compared to cells from an asynchronous population. Cell-cycle perturbations were consistent with the presence of a mitotic block after paclitaxel treatment, although changes in other cell-cycle phase fractions varied among cell lines. For experiments in combination with radiation, cytotoxicity was increased when cells were irradiated after 48 h of paclitaxel treatment but not after 8 h of treatment. CONCLUSION The duration of paclitaxel treatment and the location of cells in the cell cycle modify the degree of radiation cytotoxicity. The mechanisms of paclitaxel cytotoxicity are likely to be multifactorial because varying effects are seen in different cell lines. Furthermore, it is clear that simply increasing the number of cells in G2/M is insufficient in itself to increase the response of cells to radiation.

Isobologram analysis of X-ray--BCNU interactions in vitro.

9L rat brain tumor cells were exposed for 1 hr to either 1, 3, 5, or 7.5 μg/ml 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), followed 15 hr later with graded doses (0 to 20 Gy) of radiation. Isobolograms constructed for 1, 2, and 3 log cell kills showed that lethality from BCNU and from radiation was additive. The analysis was extended to lower levels of survival by constructing additive dose-response curves from the isobolograms. These curves define the additive, subadditive, supraadditive, and protective regions for each survival plot. For log cell kills greater than 3, additivity of lethality was found for radiation and either 1 or 7.5 μg/ml BCNU, while supra-additivity of lethality was found for radiation and either 3 or 5 μg/ml BCNU. Therefore, all concentrations of BCNU interacted positively with all doses of radiation to give enhanced cell kill.

Cytogenetic damage and the radiation-induced G1-phase checkpoint.

It is proposed that genomic integrity is preserved after DNA damage in a variety of ways. X irradiation induces a p53-dependent G1-phase cell cycle checkpoint which putatively allows time for repair of DNA damage. The p53 protein is also involved in the initiation of apoptosis after radiation-induced DNA damage, presumably leading to the elimination of lethally damaged cells from the irradiated population. To test the hypothesis that repair occurs in the additional time provided by the activation of the G1-phase checkpoint, we investigated whether the presence of a G1-phase arrest modified the frequency and type of chromosomal rearrangements at the first mitosis after irradiation. Isogenic cell lines derived from the same human glioma cell line, but differing in p53 status, were used. Purified G1-phase cells, isolated by centrifugal elutriation and X-irradiated, were studied. The wild-type p53 cell line demonstrated a dose-dependent arrest during G1 phase, as determined by flow cytometry. These cells remained in G1-phase as long as 48 h after irradiation. Cells expressing a dominant-negative p53 mutation accumulated to a much lesser extent in G1 phase after irradiation. Cells lacking the G1-phase checkpoint showed increased survival at all radiation doses. There were no significant differences in the type or frequency of total chromosomal aberrations in mitotic cells from either cell line after 1,2,4 or 6 Gy X rays, as measured by conventional cytogenetic analysis. There was an increase, however, in the number of reciprocal translocations in mitotic cells with mutant p53 (lacking a G1-phase checkpoint), as measured by fluorescence in situ hybridization with a chromosome 4-specific DNA library, but only after 6 Gy. The results suggest that the presence of a well-defined p53-dependent G1-phase arrest does not reduce chromosomal aberrations caused by low doses of ionizing radiation markedly, but may reduce the overall degree of survival by triggering other G1-phase events.

Brain Tumor Working Group Report on the 9th International Conference on Brain Tumor Research and Therapy - Organ System Program, National Cancer Institute

SummaryProceedings of the 9th International Conference on Brain Tumor Research and Therapy: 1. Introduction; 2. Surgery; 3. Radiation therapy; 4. Chemotherapy; 5. Immunotherapy; 6. Growth-regulatory alterations; 7. Molecular genetics; 8. Brain tumor invasion; 9. Normal tissue damage; 10. Polyamines.