Heat-induced changes in intracellular Na+, pH and bioenergetic status in superfused RIF-1 tumour cells determined by 23Na and 31P magnetic resonance spectroscopy.

The acute effects of hyperthermia on intracellular Na+ (Nai+), bioenergetic status and intracellular pH (pHi) were investigated in superfused Radiation Induced Fibrosarcoma-1 (RIF-1) tumour cells using shift-reagent-aided 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy. Hyperthermia at 45 degrees C for 30 min produced a 50% increase in Na, a 0.42 unit decrease in pHi and a 40-45% decrease in NTP/P(i). During post-hyperthermia superfusion at 37 degrees C, pHi and NTP/P(i) recovered to the baseline value, but Na initially decreased and then increased to the hyperthermic level 60 min after heating. Hyperthermia at 42 degrees C caused only a 15-20% increase in Nai+. In the presence of 3 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na+/H+ exchanger, the increase in Nai+ during 45 degrees C hyperthermia was attenuated, suggesting that the heat-induced increase in Nai+ was mainly due to an increase in Na+/H+ anti-porter activity. EIPA did not prevent hyperthermia-induced acidification. This suggests that pHi is controlled by other ion exchange mechanisms in addition to the Na+/H+ exchanger. EIPA increased the thermo-sensitivity of the RIF-1 tumour cells only slightly as measured by cell viability and clonogenic assays. The hyperthermia-induced irreversible increase in Nai+ suggests that changes in transmembrane ion gradients play an important role in cell damage induced by hyperthermia.     

Tumor size dependent changes in a murine fibrosarcoma: use of in vivo 31P NMR for non-invasive evaluation of tumor metabolic status

Tumor tissue contains viable hypoxic regions that are radioresistant and often chemoresistant and may therefore be responsible for some treatment failures. A subject of general interest has been the development of non-invasive means of monitoring tissue oxygen. Pulse Fourier transform 31P NMR spectroscopy can be used to estimate intracellular nucleotide triphosphates (NTP), phosphocreatinine (PCr), inorganic phosphate (Pi) and pH. We have obtained 31P NMR spectra as an indirect estimate of tissue oxygen and metabolic status in a C3H mouse fibrosarcoma FSaII. Sequential spectra were studied during tumor growth in a cohort of animals and peak area ratios for several metabolites were computed digitally by computer. During growth, tumors showed a progressive loss of PCr with increasing Pi, and most tumors greater than 250 mm3 in volume had little or no measurable PCr. The smallest tumors (38 mm3 average volume) had PCr/Pi ratios of 1.03 +/- .24, whereas tumors 250 mm3 or more had an average PCr/Pi ratio of 0.15 +/- .04. Similarly derived NTP/Pi ratios decreased with tumor size, but this change was not significant (p = .17). Radiobiologic hypoxic cell fractions were estimated using the radiation dose required to control tumor in 50% of animals (TCD50) or by the lung colony technique. Tumors less than 100 mm3 had a hypoxic cell fraction of 4% (TCD50) while tumors 250 mm3 had a 40% hypoxic cell fraction (lung colony assay). These hypoxic fraction determinations correlated well with the depletion of PCr and decline in NTP/Pi ratios seen at 250 mm3 tumor volumes. Tumor spectral changes with acute ischemia were studied after ligation of the tumor bearing limb and were similar to changes seen with tumor growth. PCr was lost within 7 minutes, with concurrent increase in Pi and loss of NTP. Complete loss of all high energy phosphates occurred by 40 minutes of occlusion. In vivo tumor 31P NMR spectroscopy can be used to estimate tissue metabolic status and may be useful in non-invasive prediction of hypoxic cell fraction, reoxygenation, and radiation treatment response.     

Effect of O6-benzylguanine analogues on sensitivity of human tumor cells to the cytotoxic effects of alkylating agents.

The effect of O6-benzylguanine, O6-(p-chlorobenzyl)guanine, and O6-(p-methylbenzyl)guanine on the sensitivity of various human tumor cell lines to alkylating agents is evaluated. The sensitivity of human colon tumor cells, HT29, to the chloroethylating agents, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 2-chloroethyl(methylsulfonyl) methanesulfonate (clomesone), and chlorozotocin was increased by pretreatment for 2 h with 25 microM of each analogue. O6-Benzylguanine was slightly more effective as a sensitizer in HT29 cells than the p-chlorobenzyl and p-methylbenzyl analogues. However, all analogues sensitized SF767 glioma cells to the cytotoxic effects of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, and clomesone to the same degree. Both cell lines were sensitized to the methylating agents streptozotocin and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide, the active intermediate of 5-(3,3-dimethyl-1-triazenyl)imidazole-4-carboxamide, by pretreatment with 10 microM O6-benzylguanine for 2 h. The number of Raji cells surviving 50 microM clomesone decreased 3-fold upon pretreatment for 2 h with 1 microM O6-benzylguanine. The degree of enhancement was dependent on the amount of alkyltransferase protein present in cell lines. For example, HT29 cells (alkyltransferase activity, 381 fmol/mg protein) exhibited a greater degree of enhancement when treated with O6-benzylguanine than SF767 (77 fmol/mg protein) and M19-MEL melanoma (36 fmol/mg protein) cells. There was no enhancement observed in mer- cell lines, U251 (less than 2 fmol/mg protein), and BE (3 fmol/mg protein), or with alkylating agents which did not produce a cytotoxic lesion at the O6 position of guanine in DNA such as cisplatin or 4-hydroperoxycyclophosphamide. Our studies suggest that O6-benzylguanine analogues may have utility in mer+ tumors as an adjuvant to a variety of alkylating agents which produce a toxic lesion at the O6 position of guanine.     

Size dependent changes in tumor phosphate metabolism after radiation therapy as detected by 31P NMR spectroscopy

In Vivo 31P NMR spectroscopy was used to study changes in phosphate metabolism that occur after irradiation of the C3H fibrosarcoma, FSaII. Previously, we have shown that small FSaII tumors (less than 250 mm3) have a greater phosphocreatinine/inorganic phosphate (PCr/Pi) ratio and a lower hypoxic cell fraction (HCF) than large FSaII tumors (greater than 250 mm3). Six small tumors (113 +/- 26 mm3) were treated with radiation doses chosen to induce local control in greater than 50% of animals, (70-100 Gy, single fraction). Minimal changes in the tumor 31P NMR spectrum were seen over eight days of monitoring. During this interval, tumor regression began a minimum of 36 hours after radiation. This contrasted with large tumors (650-1000 mm3) wherein a significant increase in the Pcr/Pi ratio was seen 44 hr after irradiation. In tumors of this size range, a tumor growth delay of 4 to 7 days is obtained after a single 70 Gy fraction of radiation. Since small FSaII tumors have a minimal HCF (approximately equal to 4%), radiation induced reoxygenation would not be expected to have a large effect on their average cellular metabolism. Large tumors of this histology have a high HCF (greater than or equal to 40%), and may therefore be expected to have a significant average change in tumor cell metabolism with reoxygenation. The 31P NMR observations of small and large tumors after irradiation are compatible with radiation induced reoxygenation in the larger tumors.     

CpG island hypermethylation of the DNA repair enzyme methyltransferase predicts response to temozolomide in primary gliomas.

PURPOSE: The DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) inhibits the killing of tumor cells by alkylating agents, and its loss in cancer cells is associated with hypermethylation of the MGMT CpG island. Thus, methylation of MGMT has been correlated with the clinical response to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in primary gliomas. Here, we investigate whether the presence of MGMT methylation in gliomas is also a good predictor of response to another emergent alkylating agent, temozolomide. EXPERIMENTAL DESIGN: Using a methylation-specific PCR approach, we assessed the methylation status of the CpG island of MGMT in 92 glioma patients who received temozolomide as first-line chemotherapy or as treatment for relapses. RESULTS: Methylation of the MGMT promoter positively correlated with the clinical response in the glioma patients receiving temozolomide as first-line chemotherapy (n = 40). Eight of 12 patients with MGMT-methylated tumors (66.7%) had a partial or complete response, compared with 7 of 28 patients with unmethylated tumors (25.0%; P = 0.030). We also found a positive association between MGMT methylation and clinical response in those patients receiving BCNU (n = 35, P = 0.041) or procarbazine/1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (n = 17, P = 0.043) as first-line chemotherapy. Overall, if we analyze the clinical response of all of the first-line chemotherapy treatments with temozolomide, BCNU, and procarbazine/1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea as a group in relation to the MGMT methylation status, MGMT hypermethylation was strongly associated with the presence of partial or complete clinical response (P < 0.001). Finally, the MGMT methylation status determined in the initial glioma tumor did not correlate with the clinical response to temozolomide when this drug was administered as treatment for relapses (P = 0.729). CONCLUSIONS: MGMT methylation predicts the clinical response of primary gliomas to first-line chemotherapy with the alkylating agent temozolomide. These results may open up possibilities for more customized treatments of human brain tumors.     

Increased therapeutic benefit through the addition of misonidazole to a nitrosourea-radiation combination

The potential therapeutic advantage of including the radiosensitizer misonidazole (MISO) in a treatment regimen combining systemic chemotherapy and localized radiotherapy was assessed in the KHT sarcoma. Tumor-bearing C3H mice were treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, administered either alone or in combination with MISO, 24 h prior to irradiating the tumors with doses ranging from 0-35 Gy. The sensitizer exposure was either a single dose of 2.5 mmol/kg or multiple injections given every 0.5 h for 8 h to maintain a sensitizer blood level of approximately 100 micrograms/ml. The chemotherapeutic agent was administered simultaneously with the single sensitizer dose or 3 h into the chronic sensitizer dosing schedule. Tumor responses to the combined modality treatments was determined using end points of tumor regrowth delay and animal tumor-free survival. Skin reactions in the treatment field were scored as a measure of normal tissue complications. MISO, administered as a single dose in the combined modality therapy, enhanced the tumor response by a factor of approximately 1.8-1.9 while increasing skin reactions approximately 1.1- to 1.2-fold. Combining 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with chronic MISO administration prior to irradiation increased the resultant tumor control probability 1.5- to 1.8-fold without enhancing normal tissue complications. Consequently using MISO as a chemopotentiator in a 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea-radiation combination can yield a significant therapeutic benefit.     

Altered O6-alkylguanine-DNA alkyltransferase activity in cell strains originating from mouse skin tumors induced by UV irradiation.

O6-Alkylguanine-DNA alkyltransferase (AGT) activity was assayed in the extracts of 47 cell strains originating from mouse skin tumors induced by UV irradiation. They were also examined for the sensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride by colony formation. The AGT activity (fmol/mg protein) of the tumor cell strains varied widely and the mean +/- SE was 72.5 +/- 9.37, while the AGT activity of the nontumor cell strains was 134 +/- 17. Among 47 strains, 6 strains showed extremely low or no AGT activity, about 5 fmol/mg protein or less, and were hypersensitive to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride. Long-term culture of the tumor cells did not change the AGT activity except in some strains which might have had coexisting normal cells in the population in early passages. All strains showed similar UV sensitivity regardless of AGT activity. This is the first report which demonstrates that about 13% of newly induced tumor cell strains are deficient in AGT activity similar to Mer-/Mex- phenotype that was found in approximately 20% of the established human tumor cell strains.     

Intratumoral administration of endostatin plasmid inhibits vascular growth and perfusion in MCa-4 murine mammary carcinomas

Endostatin, a fragment of the COOH-terminal domain of mouse collagen XVIII is a recently demonstrated endogenous inhibitor of tumor angiogenesis and endothelial cell growth. Antiangiogenic therapy with endostatin in animals requires multiple and prolonged administration of the protein. Gene therapy could provide an alternative approach to continuous local delivery of this antiangiogenic factor in vivo. Established MCa-4 murine mammary carcinomas, grown in immunodeficient mice, were treated with intratumoral injection of endostatin plasmid at 7-day intervals. At the time of sacrifice, 14 days after the first injection, endostatin-treated tumor weights were 51% of controls (P < 0.01). Tumor growth inhibition was accompanied by a marked reduction in total vascular density. Specifically, computerized image analysis showed a 18-21% increase in the median distances between tumor cells and both the nearest anatomical (CD31-stained) vessel [48.1 +/- 3.8 versus 38.3 +/- 1.6 microm (P < 0.05)] and the nearest tumor-specific (CD105-stained) vessel [48.5 +/- 1.5 versus 39.8 +/- 1.5 microm (P < 0.01)]. An increased apoptotic index of tumor cells in endostatin-treated tumors [3.2 +/- 0.5% versus 1.9 +/- 0.3% (P < 0.05)] was observed in conjunction with a significant decrease in tumor perfused vessels (DiOC7 staining), and an increase in tumor cell hypoxia (EF5 staining). Hypoxia resulting from endostatin therapy most likely caused a compensatory increase of in situ vascular endothelial growth factor (VEGF) and VEGF receptor mRNA expression. Increased immunoreactivity of endostatin staining in endostatin-treated tumors was also associated with an increased thrombospondin-1 staining [1.12 +/- 0.16 versus 2.44 +/- 0.35]. Our data suggest that intratumoral delivery of the endostatin gene efficiently suppresses murine mammary carcinoma growth and support the potential utility of the endostatin gene for cancer therapy.     

Misonidazole and radiotherapy to treat malignant glioma: A phase II trial of the radiation therapy oncology group

Fifty-four patients with Grade III or IV malignant glioma were treated with the hypoxic-cell radiosensitizer misonidazole, and radiation therapy (RT) in a Phase II trial of the Radiation Therapy Oncology Group (RTOG). The median survival was 39 weeks. Toxicity was acceptable. Almost all toxicities were reversible and self-limited. The were no significant changes in hematologic, hepatic or renal parameters. The median survival obtained is similar to the results with conventional radiotberapy. A Phase III trial is under way comparing standard radiation and BCNU chemotherapy (1,3-bis (2-chloroethyl)-1-nitrosourea) with sensitized radiation and chemotherapy (RT + misonidazole + BCNU). Additive toxicity between misonidazole and BCNU was not observed in a pilot group.     

Effects of oxygen on the metabolism of murine tumors using in vivo phosphorus-31 NMR

The effect of 100% inspired oxygen on in vivo tumor metabolism was examined using phosphorus-31 (31P) NMR spectroscopy. Isotransplants of two murine tumor histologies, designated MCaIV (C3H mammary adenocarcinoma) and FSaII (C3H fibrosarcoma), were used in syngeneic mice. Tumor volumes ranged from 30 to 1,800 mm3. Both tumor histologies are known to have a high hypoxic cell fraction when tumor volumes exceed 250 mm3. 31P nuclear magnetic resonance (NMR) spectra were obtained at 145.587 MHz, and the signal was detected using a 1.4 cm diameter, single loop coil designed to localize the signal from only the tumor. Spectral parameters for optimal signal-to-noise ratio (SNR) included a 60 degrees pulse and a 2-second recycle delay. Tumors were implanted in the hindfoot dorsum to assure that all detected mobile phosphates were of tumor origin. Phosphocreatine/inorganic phosphate (PCr/Pi) ratios of large tumors (greater than 250 mm3) were reduced compared with small tumors (less than 250 mm3) of the same histology. The increased PCr/Pi response to 100% inspired oxygen was greater for large tumors and for tumors with lower baseline PCr/Pi ratios. When host animals were given 10% oxygen for respiration, there was an increase in Pi and a decrease in both PCr and ATP. The response to 10% oxygen was observed in both large and small tumors of both tumor histologies studied. Resting skeletal muscle exhibited no alteration in the NMR spectrum during either 100 or 10% oxygen breathing. We conclude that the fractional increase in PCr/Pi ratio that occurs after 100% oxygen breathing is a sensitive, noninvasive method of detecting tumor hypoxia.     

Effect of O6-methylguanine on DNA interstrand cross-link formation by chloroethylnitrosoureas and 2-chloroethyl(methylsulfonyl)methanesulfonate

Exposure of HT29 cells in culture to O6-methylguanine is known to result in a reduction in O6-alkylguanine-DNA alkyltransferase (AGT) activity and an enhancement of sensitivity to the cytotoxic effects of chloroethylating agents. Since cytotoxicity of these agents may be mediated by the formation of interstrand cross-links, alkaline elution analysis was performed on HT29 cells treated with 1-(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and Clomesone [2-chloroethyl(methylsulfonyl)methanesulfonate] in the presence or absence of O6-methylguanine pretreatment to determine if the enhanced toxicity was due to an increase in the number of cross-links formed. Interstrand cross-linking by 1-(2-chloroethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was increased by pretreatment with 0.4 mM O6-methylguanine for 24 h. Cross-linking by Clomesone was observed only in cells exposed to 0.4 mM O6-methylguanine for 24 h prior to administration of the drug and for 12 h after administration, suggesting that the resynthesis of the AGT may prevent the cross-linking by Clomesone. Complete recovery of AGT activity after reduction to 20 to 30% of the basal level upon treatment with 0.4 mM O6-methylguanine required between 8 h and 15 h in both HT29 cells and in Raji cells which were also sensitized to 1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea by exposure to O6-methylguanine. These data suggest that the enhancement of chloroethylnitrosourea toxicity after treatment with O6-methylguanine may be related to an increase in the number of DNA cross-links and that the relatively rapid rate of AGT recovery plays a role in prevention of cross-links resulting from Clomesone.     

Chemotherapy of subcutaneous and intracranial human medulloblastoma xenografts in athymic nude mice

The continuous human medulloblastoma cell line TE-671 was grown as s.c. and intracranial xenografts in athymic nude mice. Tumor-bearing animals were treated with chemotherapeutic agents at the 10% lethal dose; s.c. xenografts were sensitive to melphalan, 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)-1-nitrosourea, and 5-azacytidine. No consistent response could be demonstrated to 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate, and no response to methylglyoxal bis(guanyl hydrazone), N-trifluoroacetyl adriamycin-14-valerate, or to 1-beta-D-arabinofuranosylcytosine was observed. Melphalan produced a significant (P = less than or equal to 0.007) increase in the median survival of mice bearing intracranial xenografts, whereas no response was seen to 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)-1-nitrosourea or 5-azacytidine. This model will allow analysis of the chemotherapeutic profile of human medulloblastoma, and provides a means to differentiate cellular sensitivity and resistance from drug access to the intracranial site.     

Potentiation of anti-cancer drug activity at low intratumoral pH induced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) and its analogue benzylguanidine (BG)

Tumour-selective acidification is of potential interest for enhanced therapeutic gain of pH sensitive drugs. In this study, we investigated the feasibility of a tumour-selective reduction of the extracellular and intracellular pH and their effect on the tumour response of selected anti-cancer drugs. In an in vitro L1210 leukaemic cell model, we confirmed enhanced cytotoxicity of chlorambucil at low extracellular pH conditions. In contrast, the alkylating drugs melphalan and cisplatin, and bioreductive agents mitomycin C and its derivative EO9, required low intracellular pH conditions for enhanced activation. Furthermore, a strong and pH-independent synergism was observed between the pH-equilibrating drug nigericin and melphalan, of which the mechanism is unclear. In radiation-induced fibrosarcoma (RIF-1) tumour-bearing mice, the extracellular pH was reduced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) or its analogue benzylguanidine (BG) plus glucose. To simultaneously reduce the intracellular pH, MIBG plus glucose were combined with the ionophore nigericin or the Na+/H+ exchanger inhibitor amiloride and the Na+-dependent HCO3-/Cl- exchanger inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). Biochemical studies confirmed an effective reduction of the extracellular pH to approximately 6.2, and anti-tumour responses to the interventions indicated a simultaneous reduction of the intracellular pH below 6.6 for at least 3 h. Combined reduction of extra- and intracellular tumour pH with melphalan increased the tumour regrowth time to 200% of the pretreatment volume from 5.7 +/- 0.6 days for melphalan alone to 8.1 +/- 0.7 days with pH manipulation (P < 0.05). Mitomycin C related tumour growth delay was enhanced by the combined interventions from 3.8 +/- 0.5 to 5.2 +/- 0.5 days (P < 0.05), but only in tumours of relatively large sizes. The interventions were non-toxic alone or in combination with the anti-cancer drugs and did not affect melphalan biodistribution. In conclusion, we have developed non-toxic interventions for sustained and selective reduction of extra- and intracellular tumour pH which potentiated the tumour responses to selected anti-cancer drugs.     

Effect of cancer on the in vivo energy state of rat liver and skeletal muscle

The effect of increasing tumor burden on host liver and skeletal muscle energy status was studied using P-31 nuclear magnetic resonance spectroscopy (NMR), in rats inoculated with a nonmetastasizing methylcholanthrene-induced sarcoma (TB), and compared to nontumor bearing (NTB) and pair-fed (PF) rats. During the 28-day study, serial measurements of body weight, food intake, and tumor volume were obtained. Using a 0.9-cm double-turn surface coil, weekly NMR measurements were obtained from liver and skeletal muscle. An increasing ratio of [Pi]/[ATP] was used as one measure of intracellular energy depletion. [Pi]/[ATP] in NTB rats remained constant over time at 0.78 +/- 0.10 in liver, and 0.30 +/- 0.10 in skeletal muscle. In TB rats, the [Pi]/[ATP] ratio increased significantly in liver (P = 0.00002) and skeletal muscle (P = 0.04) with increasing tumor burden. In PF rats, no significant change occurred in [Pi]/[ATP] in liver or skeletal muscle, indicating that declining food intake was not responsible for the change in [Pi]/[ATP] seen in TB rats. Surface-coil spectroscopy of liver and skeletal muscle permits serial measurement of visceral energy stores. Increasing tumor burden results in early, ongoing depletion of energy stores as reflected by increasing [Pi]/[ATP] in these organs.     

Formation of DNA interstrand cross-links by the novel chloroethylating agent 2-chloroethyl(methylsulfonyl)methanesulfonate: suppression by O6-alkylguanine-DNA alkyltransferase purified from human leukemic lymphoblasts

The formation of DNA interstrand cross-links was compared in DNA treated with either 1,3-bis(2-chloroethyl)-1-nitrosourea or 2-chloroethyl(methylsulfonyl)methanesulfonate. DNA that was pulse treated briefly with either of these drugs continued to form cross-links at 37 degrees C for over 8 h after drug removal, indicating that such DNA contained stable precursors of cross-links. When human O6-alkylguanine-DNA alkyltransferase was added to the drug-treated DNA further cross-link formation was prevented at all points during this protracted time course, indicating that these stable cross-link precursors also remained substrates for this repair enzyme. Although the pattern of 2-chloroethyl(methylsulfonyl)methanesulfonate-induced cross-link formation and susceptibility to suppression by O6-alkylguanine-DNA alkyltransferase resembled that for 1,3-bis(2-chloroethyl)-1-nitrosourea, quantitative differences in the rates of cross-link formation and in the amounts of O6-alkylguanine-DNA alkyltransferase required to suppress cross-link formation suggest that critical differences exist between these agents.     

Intra-arterial BCNU in the treatment of metastatic brain tumors

Thirty-one patients with metastatic brain tumors that either failed to respond or recurred after conventional therapy were treated by intra-arterial infusion of 100 mg/m2 of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) into either a carotid or vertebral artery. Five patients (three with lung cancer, one with breast cancer and one with melanoma) had a partial response of the tumor(s) in the distribution of the injected artery. In two patients, brain metastases not in the distribution of the injected artery enlarged, while the tumors perfused by the injected artery responded. In one of these patients, subsequent infusion of BCNU to the enlarging tumor resulted in a partial response. Among responders, the median survival following onset of BCNU was 17 weeks. One patient remains alive and well at 30 weeks. No permanent neurological, retinal or systemic toxicity was observed.     

Comparative effects of three nitrosourea derivatives on mammalian cell cycle progression.

Three nitrosourea analogs, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and 1-trans-(2-chloroethyl)-3-cyclohexyl-1-(4-methylcyclohexyl)-1-nitrosourea, were examined for effects on survival and cell cycle traverse capacity in exponentially growing (cycling) populations of line CHO Chinese hamster cells and in cultures arrested in G1 by isoleucine deprivation during treatment with drugs, then returned to the cycling mode by restoration of isoleucine (noncycling cells). Among parameters studied were survival, cell division, DNA initiation capacities, cell cycle distributions, and rates of cell cycle traverse in drug-treated cycling and noncycling cells utilizing a protocol combining autoradiography, cell number enumeration, and flow microfluorometry. The results obtained were in generally good agreement with results obtained in vivo in other studies and included the following. Cells treated with any of these agents accumulated preferentially in late S and G2, primarily the result of a gross increase in duration of these phases of the cell cycle. There was also a prolongation of doubling time during the early stages following drug treatment and return to the proliferating mode of cells which ultimately survived. All three drugs induced mitotic nondisjunction in cells capable of dividing and also induced polyploidy by allowing multiple rounds of progression through the cell cycle in the absence of an intervening cell division. In treated populations, the G2-arrested and polyploid cells were among the first cells to die. Treated, noncycling cells that were returned to cycle exhibited a lower survival capacity than did treated, cycling cells. Finally, 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea and 1-trans(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea induced a dramatic alteration in clonal morphology and growth patterns in surviving cells that persisted for at least a week after drug removal. The results obtained suggest that our model system may be useful as a predictive guide for determining response of susceptible tumor cells to treatment with chemotherapeutic agents.     

Requirement of the Na+/H+ exchanger for tumor growth

The Na+/H+ exchanger is involved in a variety of cellular processes, including regulation of intracellular pH and possibly the control of cell growth and proliferation. To study the role of the Na+/H+ exchanger in tumor growth, human sodium proton exchanger-deficient (HSPD) mutants were derived from the human bladder carcinoma cell line MGH-U1 (EJ) by the proton suicide selection technique (J. Pouyssegur et al., Proc. Natl. Acad. Sci. USA, 81: 4833-4837, 1984). The HSPD cells were approximately 40% larger and contained approximately 70% more DNA than the parental cells. They were unable to grow in vitro in the absence of bicarbonate at pH less than 7.0, whereas the parental cells grew well at pH greater than or equal to 6.6. This difference in acid sensitivity was abolished in the presence of bicarbonate. In contrast to the parental MGH-U1 cells, the Na+/H+-deficient HSPD cells either failed to grow tumors, or showed severely retarded tumor growth when implanted into immune-deprived mice. This difference in tumor growth was not attributed to differences in cell size and DNA content, because Na+/H+ exchange-competent large cells (HLC), derived during the same proton suicide selection process as the HSPD cells, grew tumors at a rate close to that of the parental cells. Cells derived from the few tumors which grew after implantation of HSPD mutant cells were revertants which had regained Na+/H+ activity. HSPD cells also failed to form spheroids in culture, and the only spheroid formed consisted of revertant cells which had regained both Na+/H+ exchange activity and tumorigenic capacity. These results suggest that the Na+/H+ exchanger is important for tumor growth.