Monitoring early response of experimental brain tumors to therapy using diffusion magnetic resonance imaging

Quantitative magnetic resonance imaging was performed to evaluate water diffusion and relaxation times, T1 and T2, as potential therapeutic response indicators for brain tumors using the intracranial 9L brain tumor model. Measurements were localized to a column that intersected tumor and contralateral brain and were repeated at 2-day intervals before and following a single injection of 1,3-bis(2-chloroethyl)-1-nitrosourea (13.3 mg/kg). Tumor growth was measured using T2-weighted magnetic resonance imaging to determine the volumetric tumor doubling time (Td) before (Td = 64 +/- 13 h, mean +/- SD, n = 16) and after (Td = 75 +/- 9 h, n = 4) treatment during exponential regrowth. Apparent diffusion coefficient of untreated tumors was independent of tumor volume or growth time, whereas relaxation times increased during early tumor growth. Diffusion displayed the strongest treatment effect and increased before tumor regression by 55% 6-8 days following treatment. Changes in relaxation times were also significant with increases of 16% for T1 and 27% for T2. Diffusion and relaxation times returned to pretreatment levels by 12 days after treatment. Histological examination supports the model that the observed increase in diffusion reflects an increase of extracellular space following treatment. Furthermore, the subsequent apparent diffusion coefficient decrease is a result of viable tumor cells that repopulate this space at a rate dependent on the surviving tumor cell fraction and recurrent tumor doubling time. Serial tumor volume measurements allowed determination of log cell kill of 1.0 +/- 0.3 (n = 4). These results suggest that diffusion measurements are sensitive to therapy-induced changes in cellular structure and may provide an early noninvasive indicator of treatment efficacy.     

Contributions of cell kill and posttreatment tumor growth rates to the repopulation of intracerebral 9L tumors after chemotherapy: an MRI study

The drought of progress in clinical brain tumor therapy provides an impetus for developing new treatments as well as methods for testing therapeutics in animal models. The inability of traditional assays to simultaneously measure tumor size, location, growth kinetics, and cell kill achieved by a treatment complicates the interpretation of therapy experiments in animal models. To address these issues, tumor volume measurements obtained from serial magnetic resonance images were used to noninvasively estimate cell kill values in individual rats with intracerebral 9L tumors after treatment with 0.5, 1, or 2 x LD10 doses of 1,3-bis(2-chloroethyl)-1-nitrosourea. The calculated cell kill values were consistently lower than those reported using traditional assays. A dose-dependent increase in 9L tumor doubling time after treatment was observed that significantly contributed to the time required for surviving cells to repopulate the tumor mass. This study reveals that increases in animal survival are not exclusively attributable to the fraction of tumor cells killed but rather are a function of the cell kill and repopulation kinetics, both of which vary with treatment dose.     

Treatment of human brain tumor xenografts with O6-benzyl-2'-deoxyguanosine and BCNU

O6-Methylguanine-DNA methyltransferase (MGMT), a constitutively expressed DNA repair protein, removes alkyl groups from the O6-position of guanine in DNA. Tumor cells with high MGMT activity are resistant to nitrosoureas and other agents that form toxic O6-alkyl adducts. O6-Benzylguanine (BG) inactivates the MGMT protein and thereby enhances the sensitivity of tumor cells to alkylating drugs. However, the therapeutic potential of BG is limited by its poor solubility and its nonspecific inactivation of MGMT in normal tissues as well as in tumor tissues. Consequently, BG analogues are being developed to identify agents that have more favorable pharmacological characteristics. We evaluated O6-benzyl-2'-deoxyguanosine (dBG), the 2'-deoxyribonucleoside analogue of BG, for its ability to inhibit MGMT and to potentiate 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a MGMT-positive human brain tumor xenograft, Daoy. When given i.p. 1 h before BCNU (25 mg/m2) to animals bearing s.c. tumors, dBG (134 mg/m2) produced a growth delay of 24.7 days, compared to 21.6 days after treatment with an equimolar dose of BG (90 mg/m2) plus BCNU and -0.6 days after treatment with BCNU alone. The combination of dBG + BCNU also increased the survival of animals bearing intracranial tumors by 65%. By increasing the dose of dBG to 300 mg/m2 (the maximum dose that could be delivered i.p. in a standard treatment volume), the growth delay of s.c. tumors increased from -0.1 days with BCNU alone to 39.3 days. dBG suppressed both tumor and liver MGMT activity to less than 1.5% of baseline, and dBG + BCNU induced extensive perivascular apoptosis. Because dBG is a 10-fold less potent MGMT inhibitor than BG in HT-29 cell extracts, these results illustrate the capacity of BG analogues to potentiate BCNU toxicity, despite less in vitro activity than the parent compound, and emphasize the importance of in vivo evaluation of BG analogues.     

Assessment of ganciclovir toxicity to experimental intracranial gliomas following recombinant adenoviral-mediated transfer of the herpes simplex virus thymidine kinase gene by magnetic resonance imaging and proton magnetic resonance spectroscopy

Magnetic resonance imaging and in vivo localized H magnetic resonance spectroscopy were used to evaluate a gene therapy approach for treating experimental brain tumors. This approach involved the use of an adenoviral vector to transfer the herpes simplex virus thymidine kinase (HSVtk) gene into intracerebral 9L gliosarcomas in rats followed by systemic administration of the antiherpetic agent ganciclovir. Magnetic resonance imaging quantitation of changes in intracranial 9L tumor doubling times revealed a significant variation in therapeutic response. Localized H magnetic resonance spectra of 9L tumors treated with Ad.RSVtk/ganciclovir revealed a dramatic increase in the resonance intensity at 0.9-1.3 ppm, corresponding to mobile lipids and/or lactate. Changes in intracranial tumor doubling times correlated with changes in H tumor magnetic resonance spectra, suggesting that specific changes in tumor metabolite levels may be predictive of the effectiveness of this gene therapy approach.     

Novel bifunctional anthracycline and nitrosourea chemotherapy for human bladder cancer: analysis in a preclinical survival model

A hybrid drug [N-2-chloroethylnitrosoureidodaunorubicin (AD312)] that combines structural and functional features of both anthracyclines and nitrosoureas was evaluated in a preclinical survival model of human bladder cancer. To measure the therapeutic activity of AD312, UCRU-BL13 transitional cell carcinoma cells were grown as xenografts in nude mice, and tumor growth rates were compared after i.v. administration of the drug at three dose levels. AD312 treatment at 45 and 60 mg/kg achieved 7-10-fold inhibition of tumor growth and increased host survival by 156 and 249%, respectively. Doses of 60 mg/kg showed optimal therapeutic efficacy, with sustained tumor growth inhibition, an over 2-fold increase in life span, and 40% of mice tumor free ("cured") at 120 days. Tumors were unresponsive to maximum tolerated doses of doxorubicin, a standard anthracycline used as a single agent and in combination therapies for bladder cancer. 1,3-Bis-[2-chloroethyl]-1-nitrosourea was used as a control for the apparently enhanced response of human tumors in murine hosts to nitrosoureas. 1, 3-Bis-[2-chloroethyl]-1-nitrosourea administered in three injections of 20 mg/kg did not cure mice but temporarily inhibited tumor growth by 70% and prolonged survival by 55%; its activity in this model suggests that it may be included in the repertoire of alkylating agents currently used for treatment of bladder cancers. AD312 showed increased antitumor activity with less toxicity than doxorubicin, and its bifunctional properties provide the opportunity for simultaneous treatment of individual cancer cells with two cytotoxic modalities as well as treatment of heterogeneous populations typical of bladder cancers. This novel cytotoxic drug cured doxorubicin-refractory disease and should be investigated for the clinical management of bladder cancer.     

5-[125I]iodo-2'-deoxyuridine in the radiotherapy of brain tumors in rats

Glial neoplasms of the human central nervous system have defied treatment, in part because of the limited selectivity of available cytotoxic agents. The thymidine analog 5-iodo-2'-deoxyuridine radiolabeled with the Auger electron emitter 125I (125IUdR) is highly toxic to dividing cells when it is deoxyribonucleic acid incorporated, but it is relatively innocuous when located outside the nucleus. Previous studies have shown that 125IUdR has significant antineoplastic potential against mammalian cells in vitro and direct administration of 125IUdR is effective therapy for ovarian ascites tumors in mice and neoplastic meningitis in rats. Studies using external gamma imaging and autoradiography have also shown that direct intratumoral administration of 123IUdR/125IUdR into intracerebral 9L gliosarcomas in rats results in selective uptake of the radionuclide into tumor cells. Based on these encouraging results, we have evaluated the therapeutic potential of 125IUdR in rats bearing intracerebral 9L gliosarcomas. METHODS: Iodine-125-IUdR was infused intracerebrally over a 2-day period into rats bearing 1-day-old 9L tumors and over a 6-day period into animals with 9-day-old 9L tumors; equimolar concentrations of 127IUdR were infused into control animals. Tumor growth was monitored by contrast-enhanced 1H MRI and animal survival was followed over time. RESULTS: Intracerebral tumors (3-7 mm) were readily detected by MRI. Tumor-bearing rats treated with 127IUdR succumbed within 17-24 days, whereas tumor-bearing animals treated with 125IUdR survived significantly longer, and 10%-20% of the animals were cured of tumors. CONCLUSION: These data substantiate the antineoplastic potential of 5-[125I]iodo-2'-deoxyuridine and indicate that it may be a useful agent for the therapy of solid tumors that are accessible to direct radiopharmaceutical administration.     

Correlation of dynamic contrast-enhanced magnetic resonance imaging with histologic tumor grade: comparison of macromolecular and small-molecular contrast media

BACKGROUND: The endothelial integrity of microvessels is disrupted in malignant tumors. Quantitative assays of tumor microvascular characteristics based on dynamic magnetic resonance imaging (MRI) were correlated with histopathologic grade in mammary soft tissue tumors. MATERIALS AND METHODS: A spectrum of tumors, benign through highly malignant, was induced in 33 female rats by administration of N -ethyl-N -nitrosourea (ENU), a potent carcinogen. Dynamic contrast-enhanced MRI was performed using a small-molecular contrast medium [gadopentetate, MW = 0.5 kDa] and a macromolecular contrast medium [albumin-(Gd-DTPA)30, MW = 92 kDa] at an interval of 1-2 days. Permeability surface area product (PS), as estimated by the corresponding endothelial transfer coefficient (KPS), and fractional plasma volume (fPV) were calculated for each tumor and each contrast agent using a two-compartment bi-directional kinetic model. MRI microvascular characteristics were correlated with histopathologic tumor grade. RESULTS: Tumor permeability to macromolecular contrast medium, characterized by KPS, showed a highly positive correlation with tumor grade (r 2 = 0.76, P < 10(-10)). KPS values were zero for all benign and some low-grade carcinomas, greater than zero in all other carcinomas, and increased in magnitude with higher tumor grade. A considerably smaller but significantly positive correlation was found between fPV and tumor grade using macromolecular contrast medium (r 2 = 0.25, P < 0.003). No correlation between KPS or fPV values and tumor grade was found using gadopentetate (r 2 = 0.01, P > 0.95 and r2 = 0.03, P > 0.15, respectively). CONCLUSION: Quantitative tumor microvascular permeability assays generated with macromolecular MRI contrast medium correlate closely with histologic tumor grade. No significant correlation is found using small-molecular gadopentetate.     

Correlation of chloroethylnitrosourea resistance with ERCC-2 expression in human tumor cell lines as determined by quantitative competitive polymerase chain reaction

We have developed a method to quantitate ERCC-2 gene expression in tumor cell lines. A mutant ERCC-2 DNA fragment (1-bp mutation) is used as a competitive DNA template in a coamplification PCR reaction with cDNA obtained by reverse transcribing DNase-free total RNA from six human tumor cell lines. The PCR products are separated on agarose gel by virtue of their differential banding pattern upon restriction enzyme digestion. Densitometric readings of the PCR products from a negative film of the gel are used to establish a linear regression curve, which in turn is used to quantitate ERCC-2 levels. Beta-actin expression is similarly quantitated. Normalized ERCC-2 gene expression (either to beta-actin or to total RNA) correlates with cytotoxicity of 1,3-bis-(2-chloroethyl)-1-nitrosourea or (2-chloroethyl)-3-sarcosinamide-1-nitrosourea, suggesting that ERCC-2 may play an important role in drug resistance in these cell lines. This method is reliable and can be used to quantitate gene expression in clinical tumor specimens.     

Induction of specific-locus and dominant lethal mutations in male mice by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)

1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) induced dominant lethal and specific-locus mutations in male mice. For both compounds the germ cell stage sensitive to the induction of dominant lethal mutations was dose dependent. A dose of 5 mg BCNU per kg b.wt. induced dominant lethal mutations primarily in spermatocytes, whereas higher doses of BCNU induced dominant lethals in spermatids and spermatocytes. Following doses of 5 and 10 mg CCNU per kg b.wt. dominant lethals were induced in spermatids and spermatocytes similar to the results for higher doses of BCNU. Higher dose exposure to BCNU and CCNU was associated with dominant lethals expressed as pre-implantation loss (reduction in total number of implants). In addition, higher doses of CCNU showed a cytotoxic effect in differentiating spermatogonia. Both compounds induced specific-locus mutations in post-spermatogonial germ cell stages of mice. However, CCNU increased also the specific-locus mutation frequency in spermatogonia in two out of three experiments. We conclude in analogy with criteria developed by IARC, that BCNU and CCNU are potential human mutagens.     

Methylator resistance mediated by mismatch repair deficiency in a glioblastoma multiforme xenograft

A methylator-resistant human glioblastoma multiforme xenograft, D-245 MG (PR), in athymic nude mice was established by serially treating the parent xenograft D-245 MG with procarbazine. D-245 MG xenografts were sensitive to procarbazine, temozolomide, N-methyl-N-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, 9-aminocamptothecin, topotecan, CPT-11, cyclophosphamide, and busulfan. D-245 MG (PR) xenografts were resistant to procarbazine, temozolomide, N-methyl-N-nitrosourea, and busulfan, but they were sensitive to the other agents. Both D-245 MG and D-245 MG (PR) xenografts displayed no O6-alkylguanine-DNA alkyltransferase activity, and their levels of glutathione and glutathione-S-transferase were similar. D-245 MG xenografts expressed the human mismatch repair proteins hMSH2 and hMLH1, whereas D-245 MG (PR) expressed hMLH1 but not hMSH2.     

Activity of fotemustine in medulloblastoma and malignant glioma xenografts in relation to O6-alkylguanine-DNA alkyltransferase and alkylpurine-DNA N-glycosylase activity

Fotemustine is a chloroethylnitrosourea with antitumor activity in disseminated melanoma and adult primary brain tumors. Because new drugs are required for the treatment of medulloblastoma in children, we evaluated the preclinical antitumor activity of fotemustine in four s.c. medulloblastoma xenografts, in comparison with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Both drugs were administered as a single i.p. injection to nude mice bearing advanced-stage tumor. Fotemustine displayed significant antitumor activity in three of four medulloblastoma xenografts; two, IGRM34 and IGRM57, were highly sensitive, with 37 and 100% tumor-free survivors, respectively, more than 120 days after treatment at the highest nontoxic dose (50 mg/kg). Fotemustine was also highly active in a malignant glioma xenograft (IGRG88; five of six tumor-free survivors on day 177). Fotemustine proved to be significantly more active than BCNU in IGRM34 and the glioma xenograft IGRG88. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) was detected in all tumor xenografts, ranging in activity from 6 to 892 fmol/mg protein. The high in vivo sensitivity to fotemustine and BCNU observed in three xenografts was clearly associated with a low ATase activity (> 20 fmol/mg), whereas the two poorly sensitive or refractory medulloblastoma xenografts showed high ATase activity (> 500 fmol/mg). Alkylpurine-DNA N-glycosylase activity was detected in all tumor xenografts but at levels ranging only from 513 to 1105 fmol/mg/h; no consistent relationship was found between alkylpurine-DNA N-glycosylase activity and the in vivo sensitivity to the two chloroethylnitrosoureas. The improved activity and tolerance of fotemustine in comparison with BCNU in pediatric medulloblastoma xenografts strongly support the clinical development of this agent in children with brain tumors, in which ATase should be examined as a potential prognostic indicator.     

IL-10 gene transfer to intracranial 9L glioma: tumor inhibition and cooperation with IL-2

This study examines the effects of interleukin-10 (IL-10) and combination IL-10 + IL-2 gene transfer on experimental brain tumor growth in vivo. 9L gliosarcoma cells were engineered to stably express murine IL-10 (9L-IL-10 cells) and implanted subcutaneously or to the caudate/putamen of syngeneic rats. The growth of tumors expressing IL-10 was substantially reduced compared to that of control tumors (p < 0.05). Intracranial tumors expressing IL-10 and IL-2 were established by co-implanting 9L-IL-10 cells with endothelial cells engineered to express IL-2. At 14 days post-implantation, tumors expressing IL-10 + IL-2 were 99% smaller than control-transfected tumors (p < 0.0001). This extent of anti-tumor effect could not be achieved by expression of IL-10 or IL-2 alone within tumors. Neither IL-10 nor a combination of IL-10 + IL-2 gene delivery inhibited tumor growth in severe combined immunodeficient (SCID-Beige) mice (p > 0.05). Immunohistochemical analysis revealed that IL-10 + IL-2 gene delivery markedly increased T-cell infiltration within the striatum ipsilateral to tumor cell implantation. These findings establish that IL-10 expression, particularly in combination with IL-2 expression, can have significant immune-dependent anti-tumor actions within intracranial gliomas.     

Retroviral transfer of a bacterial alkyltransferase gene (ada) into human bone marrow cells protects against O6-benzylguanine plus 1, 3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity

The antitumor activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is limited by the O6-alkylguanine-DNA alkyltransferase (ATase) in tumor cells and by delayed myelosuppression. Inactivation of neoplastic ATase by O6-benzylguanine (BG) improves the therapeutic index for BCNU. We have demonstrated previously that BG + BCNU-induced myelosuppression in mice is reduced by expression of the BG-resistant ATase ada in murine bone marrow. We have now generated an amphotropic retrovirus containing the ada gene and tested the effectiveness of ada expression in preventing BG + BCNU cytotoxicity in human hematopoietic progenitor cells. A retroviral producer clone with a biological titer of 6.5 x 10(4) colony-forming units/ml and 4.4 pmol ATase/mg protein was used for transduction of bone marrow. Cocultivation of these ada producer cells with progenitor cells from six normal individuals resulted in 1.9-3. 9-fold protection against BG + BCNU-induced cytotoxicity in committed progenitor cell assays. Furthermore, this cytoprotective effect was associated with a high transduction efficiency (40%) and a 2-fold increase of ATase activity in the surviving committed progenitor cell colonies. These data provide a basis for testing the clinical effectiveness of retroviral ada gene transfer into hematopoietic cells to increase the therapeutic index of BG + BCNU.     

Enhanced antitumor efficacy of cisplatin in combination with ALRT1057 (9-cis retinoic acid) in human oral squamous carcinoma xenografts in nude mice

Cisplatin (DDP) is commonly used to treat head and neck tumors. Therapy frequently fails due to development of DDP resistance or toxicities associated with DDP therapy. In this study, effects of ALRT1057 [9-cis retinoic acid (9-cis RA)] on DDP cytotoxicity were studied in a human oral squamous carcinoma xenograft model. Mice bearing xenografts were dosed p.o. daily 5 days/week with 30 mg/kg 9-cis RA and/or i.p. twice weekly with 0.3-0.9 mg/kg DDP. Maximum tolerated doses of 9-cis RA and DDP were approximately 60 and >/=2.9 mg/kg, respectively, under their dosing schedules and routes of administration. Control tumors grew rapidly with mean doubling times of 4 +/- 1 days and reached mean volumes of 1982 +/- 199 (SE) mm3 after 24 days. DDP at doses of 0.3, 0.45, and 0.9 mg/kg inhibited tumor growth by 28, 47, and 86%, respectively, 24 days after tumor cell implantation. Thirty mg/kg 9-cis RA inhibited tumor growth by 25%. In combination, 0.3 mg/kg DDP + 30 mg/kg 9-cis RA inhibited tumor growth by 68%; 0.45 mg/kg DDP + 30 mg/kg 9-cis RA inhibited growth by 78%. These decreases were greater than those that would have been produced by either agent summed separately. Of importance, at doses of 9-cis RA that enhanced DDP cytotoxicity, no change in dose tolerance was observed as compared to tolerances observed for either agent alone, indicating that 9-cis RA increased sensitivity to DDP without altering systemic toxicity. In addition, 9-cis RA profoundly altered squamous cell carcinoma phenotypes by suppressing squamous cell differentiation, resulting in tumors with increased numbers of basal cells. In contrast, DDP selectively depleted proliferating basal cells from carcinomas. In combination, morphological changes produced by 9-cis RA alone predominated, suggesting a possible basis for enhanced DDP sensitivity in tumors exposed to both agents. These data demonstrate that 9-cis RA enhances tumor sensitivity to DDP, and suggest that this combination should be tested in Phase I-II clinical trials for its potential for improving anticancer therapy of squamous cell cancers.     

Female fifteen-spined sticklebacks prefer better fathers

PURPOSE: Due to the cytotoxicity of DNA-bound iodine-125, 5-[125I]Iodo-2'-deoxyuridine ([125I]IUdR), an analog of thymidine, has long been recognized as possessing therapeutic potential. In this work, the feasibility and potential effectiveness of hepatic artery infusion of [125I]IUdR is examined. METHODS: A mathematical model has been developed that simulates tumor growth and response to [125I]IUdR treatment. The model is used to examine the efficacy and potential toxicity of prolonged infusion therapy. Treatment of kinetically homogeneous tumors with potential doubling times of either 4, 5, or 6 days is simulated. Assuming uniformly distributed activity, absorbed dose estimates to the red marrow, liver and whole-body are calculated to assess the potential toxicity of treatment. RESULTS: Nine to 10 logs of tumor-cell kill over a 7- to 20-day period are predicted by the various simulations examined. The most slowly proliferating tumor was also the most difficult to eradicate. During the infusion time, tumor-cell loss consisted of two components: A plateau phase, beginning at the start of infusion and ending once the infusion time exceeded the potential doubling time of the tumor; and a rapid cell-reduction phase that was close to log-linear. Beyond the plateau phase, treatment efficacy was highly sensitive to tumor activity concentration. CONCLUSIONS: Model predictions suggest that [125I]IUdR will be highly dependent upon the potential doubling time of the tumor. Significant tumor cell kill will require infusion durations that exceed the longest potential doubling time in the tumor-cell population.     

Automatic tumor segmentation using knowledge-based techniques

A system that automatically segments and labels glioblastoma-multiforme tumors in magnetic resonance images (MRI's) of the human brain is presented. The MRI's consist of T1-weighted, proton density, and T2-weighted feature images and are processed by a system which integrates knowledge-based (KB) techniques with multispectral analysis. Initial segmentation is performed by an unsupervised clustering algorithm. The segmented image, along with cluster centers for each class are provided to a rule-based expert system which extracts the intracranial region. Multispectral histogram analysis separates suspected tumor from the rest of the intracranial region, with region analysis used in performing the final tumor labeling. This system has been trained on three volume data sets and tested on thirteen unseen volume data sets acquired from a single MRI system. The KB tumor segmentation was compared with supervised, radiologist-labeled "ground truth" tumor volumes and supervised k-nearest neighbors tumor segmentations. The results of this system generally correspond well to ground truth, both on a per slice basis and more importantly in tracking total tumor volume during treatment over time.     

Comparison of gadopentetate dimeglumine and albumin-(Gd-DTPA)30 for microvessel characterization in an intracranial glioma model

To compare the performance of macromolecular albumin gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA)30 and low molecular weight gadopentetate dimeglumine for microvessel characterization, we examined an intracranial 9L glioma model in which increased angiogenesis, hypervascularity, and hyperpermeability mimic characteristics of clinical malignant brain tumors. Dynamic MRI data were analyzed using a bidirectional, two-compartment kinetic model to extract quantitative estimates for fractional blood volume (fBV) and permeability surface area product (PS). Three criteria were used for comparison of contrast agent performance: (a) tumor conspicuity, defined as the contrast-to-noise ratio (CNR); (b) dynamic range of differential permeability estimates between tumor and normal brain; (c) reasonableness of blood volume estimates. Gadopentetate was superior to macromolecular albumin-(Gd-DTPA)30 for detection of 9L brain gliomas and for measurements of hyperpermeability.     

Active site specific inactivation of chymotrypsin by cyclohexyl isocyanate formed during degradation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea

Prolonged incubation of 1-(2-chloroethyl)-3-([1-14C]cyclohexyl)-1-nitrosourea with chymotrypsin resulted in covalent modification and concomitant inactivation of chymotrypsin via degradation of the nitrosourea to form cyclohexyl isocyanate. Cyclohexyl isocyanate was shown to be an active-site-specific inactivator of chymotrypsin. A cyclohexyl isocyanate to enzyme molar ratio of 0.63 was required to produce 50% enzyme inactivation, thus demonstrating the high specificity of inactivation. At 2.38 X 10(-4) M chymotrypsin this near stoichiometric inactivation was not significantly affected by the presence of 1, 5, and 10 mM L-lysine. Degradation of an excess of 1-(2-chloroethyl)-3-([1-14C]-cyclohexyl)-1-nitrosourea in the presence of enzyme yielded 1.11 +/- 0.07 mol of covalently bound [14C]cyclohexyl moiety per mol of enzyme inactivated. Short-term incubation demonstrated that the nitrosourea neither inhibited nor protected the enzyme from cyclohexyl isocyanate inactivation. Treatment of chymotrypsin with less than stoichiometric amounts of cyclohexyl isocyanate or titration of the active-site serine with phenylmethanesulfonyl fluoride followed by in situ degradation of excess 1-(2-chloroethyl)-3-([1-14C]cyclohexyl)-1-nitrosourea resulted in a decreased amount of covalently bound 14C proportional to the extent of inactivation by these reagents prior to 14C labeling. These results strongly suggest that cyclohexyl isocyanate, whether added directly or generated by CCNU degradation, reacted almost exclusively with the active site of the enzyme. The extent of this inactivation indicates that 70% of the CCNU degraded in such a manner as to form cyclohexyl isocyanate.     

A placebo-controlled crossover study of rizatriptan in the treatment of multiple migraine attacks. Rizatriptan Multiple Attack Study Group

Recent data have suggested that mitochondria play a supportive role in maintaining the tumorigenic phenotype. Indeed, antimitochondrial agents have been hypothesized to be potential chemosensitizers to human malignancy. We assessed the utility of this approach by characterizing the antimitochondrial activity of 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (AG17), in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in two human glioblastoma cell lines. AG17 (NSC 242557) is a tyrphostin that has been thought to have some antimitochondrial activity, with limited tyrosine kinase antagonism, and was used at noncytotoxic and nongrowth-inhibitory concentrations (0.25 microM). Glioblastoma cells were incubated in AG17, and changes in mitochondrial activity were determined. Tumor cells became auxotrophically dependent on uridine and pyruvate, indicating the lack of a functioning respiratory chain. Despite this, cells continued to exhibit no growth-inhibitory effects. Exposure to AG17 was associated with significant depolarization of the mitochondrial membrane potential and decreases in mitochondrial mass in both glioblastoma cell lines, correlating with the finding of auxotrophic dependence. In contrast, normal human astrocytes treated with the same dose of AG17 did not show changes in growth, mitochondrial membrane potential, or mass. Indeed, auxotrophic dependence on uridine and pyruvate could not be established in these cells. Glioblastoma cells became significantly more responsive to BCNU chemotherapy with AG17 pretreatment; a linear relationship was noted that correlated the number as well as percentage of polarized mitochondria with glioblastoma cell survival at the highest dose of BCNU used (144 microg/ml). Normal human astrocytes did not change with regard to the dose response to BCNU with previous incubation with AG17. No difference was found in the type of cellular death (apoptosis) in either of the glioblastoma cell lines, with BCNU treatment alone, or with the combination AG17 and BCNU, despite the decrease in polarized mitochondria and mitochondrial mass. AG17 has antimitochondrial properties when used at low dose in human glioblastoma, which are relatively specific to tumor cells when compared with normal astrocytes. The use of AG17 as a chemosensitizer, with drugs such as BCNU, offers a new and possibly effective approach to be developed in patients with glial tumors.     

Novel myofilament Ca2+-sensitizing property of xanthine oxidase inhibitors

To better understand the relationship of the growth characteristics of tumor tissues and their response to ionizing radiation alone and in combination with local tumor hyperthermia, we compared three different tumor sublines of the Dunning rat prostate carcinoma R3327. This report includes results obtained with the anaplastic AT1 subline (volume doubling time 5.2 days), the moderately differentiated mucin-secreting HI subline (volume doubling time about 9 days) and the well-differentiated, hormone-dependent H subline (volume doubling time about 17 days). The effects of single doses of photons (10 to 40 Gy) with and without local tumor hyperthermia (35 min immersion at 43.5 degrees C) were quantified by growth delay. The time to reach five times the volume at the time of treatment after 30 Gy alone was found to be 56.0, 134.9 and 184.0 days for the R3327-AT1, HI and H tumors, respectively. The R3327-H tumor was more radiosensitive than the AT1 or HI subline. Five of nine R3327-H tumors were controlled locally with a single dose of photons (40 Gy). Local tumor hyperthermia alone induced growth delay in both differentiated tumors, while the anaplastic tumor subline did not respond. Combined treatment modalities with heat applied directly after irradiation revealed isoeffective thermal enhancement ratios for 30 Gy which decreased from 1.59 for the AT1 tumor and 1.42 for the HI tumor to 1.23 in the well-differentiated subline R3327-H.