Anemic stress as a trigger of myelogenous leukemia in the unirradiated RF mouse.

Ninety-six percent of mice that were bled of 50 percent of their blood volume when they were 9 weeks old succumbed to myelogenous leukemia by 15 months after phlebotomy, the majority of them dying between 7 and 10 months after this treatment. These results suggest that (i) anemia is an effective stress for triggering myelogenous leukemia in animals that are predisposed to the disease, (ii) the RF mouse is "naturally" prone to the development of myelogenous leukemia, and (iii) the concept of two-step de novo induction of myelogenous leukemia appears to be applicable in this animal.

Growth kinetics of mammary tumor 13762 in rats previously cured by chemotherapy.

We studied the growth capabilities of mammary tumor 13762 transplanted into inbred F344 rats previously cured of tumors by cell kinetically based sequential chemotherapy. Of the 18 challenge tumors, 4 were completely rejected, and nonrejected tumors grew at subnormal rates. The subnormal growth was specific for the cured rats because tumor growth in age- and therapy-matched non-tumor-bearing controls was normal. Cell kinetic studies with the use of in vitro techniques for the [3H]dThd labeling index, DNA synthesis time, and primer-dependent DNA polymerase labeling index (an in vitro estimate of growth fraction) indicated that the subnormal growth rates of the 13762 tumor in cured rats were due to subnormal tumor cell production. Cell loss rates were similar in tumors growing in cured rats and in size-matched tumors growing in normal controls. The results are consistent with the possibility that the subnormal growth of 13762 challenge tumors in chemotherapeutically cured F344 rats was mediated by immune factors.

Cell kinetics in vivo and in vitro for C3H/He spontaneous mammary tumors.

Cell kinetics in spontaneous C3H/HeJ mammary tumors of retired-breeder mice was studied by in vivo and in vitro techniques. The [3H]TdR labeling index (LI), the DNA synthesis time (TS), and the primer-dependent DNA polymerase assay LI [an in vitro estimate of tumor growth fraction (GF)] were compared to similar measurements made in vivo. These measurements as well as the calculated cell kinetic parameters derived from these data were not different in in vivo and in vitro studies. Furthermore, the cell kinetic parameters in tumors classified histologically as type A or type B mammary tumors were also similar. Although considerable variation in volume doubling times (Td's), [3H]TdR LI's, potential doubling times, cell cycle times (Tc's), and cell loss was found, Ts's were similar in all mammary tumors. No correlation between tumor volume or tumor weight and cell kinetic parameters was seen. However, the most slowly growing tumors (i.e., tumors with the longest Td's) tended to have the lowest [3H]TdR LI's, the longest Tc's, and the highest cell loss factors. No correlation was found between the GF and Td. However, tumors with the most rapidly proliferating cell populations tended to have the highest GF's.

Acute hemorrhagic necrosis of tumors induced by interleukin-1 alpha: effects independent of tumor necrosis factor.

Tumor necrosis factor (TNF), a protein synthesized in response to the endotoxin bacterial lipopolysaccharide (LPS), is the classical mediator of acute hemorrhagic necrosis of tumors. We have demonstrated that interleukin-1 alpha (IL-1 alpha), with a spectrum of activities very similar to those of TNF, also causes acute hemorrhagic necrosis of tumors. Both TNF and IL-1 induce a cascade of events including the synthesis or release of each other. The present studies were thus undertaken to determine whether the hemorrhagic necrosis induced in tumors by IL-1 alpha is due to TNF. Kinetic parameters of IL-1 alpha-induced hemorrhage were similar to those observed with recombinant murine TNF-alpha (TNF-alpha) or LPS in RIF-1 fibrosarcomas in C3H/HeN (endotoxin-sensitive) mice. However, the amount of TNF found in the sera or tumors of animals treated with LPS was more than 20-fold higher than in mice treated with IL-1 alpha, and LPS induced similar degrees of hemorrhagic necrosis, which was measured by determining the packed volume of red blood cells by 59Fe labeling. A low but significantly hemorrhagic dose of IL-1 alpha induced no detectable TNF in tumors. Pretreatment with 250 micrograms of neutralizing antibody to TNF had no effect on IL-1 alpha-induced hemorrhage, whereas TNF-alpha- and LPS-induced hemorrhagic effects were significantly reduced. These results demonstrate an important antitumor activity of IL-1 alpha that appears to be independent of TNF.

Effect of cyclophosphamide on the pathophysiology of RIF-1 solid tumors.

The present experiments were conducted to determine the effects of cyclophosphamide (150 mg/kg) on the pathophysiology of RIF-1 solid tumors and to determine the temporal relationship between treatment mediated changes in tumor vascular physiology, cell proliferation, and chemoresponsiveness in vivo. Capillary permeability and plasma and extracellular water volumes were determined by a 125I-bovine serum albumin, 51Cr-EDTA double isotope dilution assay at various intervals after cyclophosphamide. Tumor blood flow and exchangeable erythrocyte vascular volumes were determined by 86RbCl distribution and 51Cr-labeled erythrocyte dilution methods. Cell proliferation in RIF-1 tumors, assessed by [3H]thymidine labeling index and tumor growth fraction (primer-dependent DNA polymerase labeling assay) measurements, was inhibited for up to 3 days by cyclophosphamide. Although tumor regrowth was not apparent until Day 10, cell kinetic studies indicated proliferative recovery in the surviving cell population on Days 4 and 5 after treatment. Increases in tumor blood flow and tumor vascular volumes were temporally coincident with this proliferative response. In split-dose experiments, the time-dependent increases in the chemoresponsiveness of RIF-1 tumors, after cyclophosphamide, may be due not only to the increased proliferation of repopulating cells, but also to vascular responses attendant with cytoreduction.

Antiproliferative effects of corticosteroids in C3H/HeJ Mammary tumors and implications for sequential combination chemotherapy.

Corticosteroid-induced inhibition of cell proliferation and tumor growth was studied in first-generation transplants (FGMT) of spontaneous C3H/HeJ mammary tumors (SMT). Competitive binding studies using the dextran-coated charcoal method demonstrated that both SMT and FGMT exhibit high-affinity, low-capacity cytoplasmic binding sites for corticosteroids. Free cytoplasmic binding sites, determined by Scatchard analysis of dexamethasone (DEX) binding data, were more abundant in SMT (323 +/- 45 fmol/mg) than in FGMT (199 +/- 35 fmol/mg). Apparent dissociation constants, 3.83 +/- 1.14 and 5.06 +/- 1.53 nM for SMT and FGMT, respectively, were consistent with those found in other corticosteroid-sensitive tissues. In vivo treatment of FGMT with DEX or methylprednisolone resulted in dose-dependent inhibition of cell proliferation and tumor growth. The recovery kinetics after three doses of either methylprednisolone or DEX (10 mg/kg every 12 hr) suggested reversible G1 progression delay. Changes in the [3H]thymidine labeling index after steroid treatment indicated maximal S-phase cellularity 18 to 24 hr after methylprednisolone and 42 to 48 hr after DEX. On the basis of regrowth delay measurements, the effectiveness of sequential therapy with corticosteroids and either 5-fluorouracil or especially vincristine was seen to be time sequence dependent. The most effective intervals were those in which vincristine and/or 5-fluorouracil was given to coincide with maximal S-phase cellularity after steroid treatments.

Effect of dexamethasone on vascular function in RIF-1 tumors.

The present series of experiments was conducted to determine the effect of dexamethasone on vascular function and cell proliferation in s.c. RIF-1 tumors. 125I-BSA, 51Cr-EDTA dilution techniques were used to evaluate dexamethasone induced changes in tumor plasma water, capillary permeability, and extracellular water volumes, while 59Fe and 51Cr labeled erythrocyte techniques were used to assess changes in tumor exchangeable erythrocyte volumes. 86RbCl distribution studies were also conducted to evaluate vascular perfusion in RIF-1 tumors after dexamethasone treatment. In this corticosteroid receptor containing tumor model, dexamethasone had profound effects on all of the measured parameters of vascular function. Reduced tumor cell proliferation after dexamethasone treatments was accompanied by reduced capillary permeability, reduced interstitial water volumes, increased plasma volumes, and reduced vascular perfusion. Serial studies after dexamethasone treatments indicated that increases in vascular perfusion preceded proliferative recovery. Intervals of maximal [3H]thymidine labeling after dexamethasone were characterized by transient increases in capillary permeability, interstitial water volumes, and tumor erythrocyte exchange with the general circulation. At intervals of maximal cell proliferation (36-48 h after dex) 86RbCl distribution in tumors was about 3 times that seen in untreated controls. The results seem to indicate that, as in edematous normal tissues, dexamethasone can have profound effects on vascular function and water compartmentalization in RIF-1 tumors.

Cell kinetic-directed sequential chemotherapy with cyclophosphamide and adriamycin in T1699 mammary tumors.

The present studies were initiated to investigate the changes [3H]deoxythymidine labeling index, primer-dependent DNA polymerase labeling index, and S-G2 transition after treatment of T1699 transplantable mouse mammary tumors with Adriamycin (5 mg/kg) and cyclophosphamide (100 mg/kg). Treatment with these agents resulted in intervals of subnormal tumor cell proliferation as indicated by decreased [3H]deoxythymidine labeling index, primer-dependent DNA polymerase labeling index, and S-G2 transition. Recovery, as indicated by increases in [3H]deoxythymidine labeling index, primer-dependent DNA polymerase labeling index, and S-G2 transition, was observed three days after Adriamycin treatment and six to seven days after cyclophosphamide treatment. To evaluate the predictive nature of the kinetic changes for effective time sequencing, sequential combination chemotherapy protocols were designed and tested in T1699 tumor-bearing mice. The results from these studies showed that the most effective chemotherapy schedules were those in which the drugs were sequenced to coincide with the cell kinetic recovery from the single agents alone. These effective sequencing intervals were also found to be effective when used in multifraction sequential combination chemotherapy protocols. The results suggest that changes in cell kinetic parameters following drug perturbation can provide indications as to potentially efficacious as well as nonefficacious sequencing intervals.

Effect of methylprednisolone on the cell kinetic response of C3H/HeJ mammary tumors to cyclophosphamide and adriamycin.

The effect of methylprednisolone (MP) on the cell kinetic response to cyclophosphamide (CP) and Adriamycin (ADR) in C3H/HeJ spontaneous mammary tumors and hematopoietic tissue was investigated. The [3H]deoxythymidine labelingg index, the primer-dependent DNA polymerase labeling index (an estimate of tumor growth fraction), and the mitotic index were determined at various intervals after treatment. Treatment consisted of CP (200 mg/kg) on Day 0 plus ADR (2 mg/kg) on Day 1 with or without MP every 12 hr for 9 doses beginning on Day 2. In tumors treated with CP and ADR alone, changes in the kinetic parameters suggested proliferative recovery between Days 3 and 4 which coincided with bone marrow recovery. In tumors treated with CP, ADR, and MP, although the timing of the hematopoietic recovery was not affected by MP, the overshoot of the [3H]deoxythymidine labelin index on Days 3 and 4 was abolished. Proliferative recovery in the tumor was delayed until after cessation of MP treatments. Cell kinetic changes in the tumor after CP, ADR, and MP were used to design effective sequential chemotherapy which obviated the hematopoietic toxicity associated with sequential therapy designed from cell kinetic changes after CP and ADR alone.

Receptor-dependent antiproliferative effects of corticosteroids in radiation-induced fibrosarcomas and implications for sequential therapy.

Competitive binding studies with [3H]dexamethasone and Scatchard analysis demonstrated a single class of high-affinity, low-capacity glucocorticoid receptor sites in 105,000 x g cytosols from radiation-induced fibrosarcomas. In vivo, both dexamethasone (DEX) and methylprednisolone treatments resulted in dose-dependent inhibition of tumor growth and cell proliferation. Changes in the sensitivity of the clonogenic cell population to 3 mM hydroxyurea were used to assess changes in the clonogenic cell proliferation during and after treatments with DEX or methylprednisolone. Neither methylprednisolone nor DEX given every 12 hr for three doses resulted in significant cell kill in the clonogenic fraction. However, changes in the hydroxyurea sensitivity of the clonogenic population after cessation of DEX treatments indicated G1 cell cycle progression delay with transient enrichment of S-phase clonogenic cells 24 to 48 hr after cessation of DEX treatments. The duration of the DEX-induced progression delay and the timing of maximal S-phase cellularity after DEX was directly correlated with the level of glucocorticoid receptors in the treated tumors. Using regrowth delay to assess the efficacy of kinetically directed sequential chemotherapy, the effectiveness of vincristine, given after DEX, was highly sequence dependent, with the most effective treatment interval being coincident with maximal S-phase clonogenic fraction. Other studies indicated that the effectiveness of cyclophosphamide could also be increased by time sequencing after DEX.

Correlation between glucocorticoid receptor content and the antiproliferative effect of dexamethasone in experimental solid tumors.

The present studies were conducted to assess the antiproliferative effects of dexamethasone (DEX) in murine, rat, and xenograft tumor models and to determine if this kinetic response could be correlated with the level of cytosolic glucocorticoid receptors. Saturable DEX receptors were determined by the dextran-coated charcoal competitive-binding assay, and the antiproliferative effects of DEX were determined by serial measurements of [3H]thymidine labeling indices before and after DEX treatments. The results from such studies in 3 colon tumor lines, one mouse (CO-38) and 2 human xenograft lines (LoVo, H81-4), were qualitatively similar. [3H]Thymidine labeling indices, initially reduced 50 to 60% by the DEX treatments, subsequently increased to as much as 2 times control values, 24 to 36 hr later. With the R3230AC rat mammary tumor model, the DEX dose-dependent antiproliferative effect was similar regardless of whether the tumor was grown in its syngeneic host, Fischer 344 rats, or as a xenograft in athymic nude mice. Studies using the B16 melanoma and SaD2 sarcoma tumor models indicated that the in vivo efficacy of vincristine, given after DEX, was highly sequence dependent. The results from these and other studies in glucocorticoid receptor-positive solid tumor models indicated that the DEX dose-dependent antiproliferative effect and the timing for maximal post-DEX [3H]thymidine labeling indices can be directly correlated with the level of saturable glucocorticoid receptors.

Role of corticosteroid hormones in the control of cell proliferation in residual tumor after surgical cytoreduction.

Changes in tumor cell proliferation in local and distant residual tumor were studied after subtotal surgical cytoreduction in three experimental tumor models varying in corticosteroid receptor content, cell proliferation, and animal host. In residual s.c. RIF-1 anf R3230AC tumors, proliferation was inhibited within 24 hr after 75% resection. Subsequently, intervals of increased proliferation, characterized by increases in tritiated thymidine [( 3H]-dThd) labeling index, primer-dependent DNA polymerase labeling indices, and S-phase clonogenic fractions, were observed. In RIF-1 "artificial" lung metastases. [3H]dThd uptake in tumor-bearing lungs increased by about 70% at 3 days after amputation of "primary" tumor-bearing legs. When dexamethasone was given every 12 hr during the postsurgical recovery interval, changes in [3H]dThd labeling indices and [3H]dThd uptake per lung indicated that the proliferative recovery was delayed until after cessation of dexamethasone treatments. Other studies with RIF-1 tumors indicated that postsurgical tumors indicated that postsurgical proliferation inhibition was dependent on intact adrenal function and that the initiation of postsurgical proliferative recovery was preceded by reestablishment of normal serum corticosterone levels and presurgical levels of saturable glucocorticosteroid receptor. The effectiveness of cyclophosphamide 5-fluorouracil after surgery was time dependent in residual local and distant tumor, with the most efficacious intervals being coincident with postsurgical proliferative recovery. Our results indicate that, in these experimental tumor models, changes in endogenous corticosteroid hormones resulting from the surgical trauma, cellular corticosteroid hormone receptor levels and cytoreduction may influence the time course of the proliferative response in residual tumor after surgical cytoreduction.

Adriamycin-induced delayed erythropoietic injury expressed following anemia stress.

The present studies were undertaken to compare anemia-induced erythropoietic responses in femoral marrows and spleens of mice pretreated with Adriamycin (ADR) or 1-beta-D-arabinofuranosylcytosine with those of untreated age-matched controls. Mice were bled 45 or 120 days after drug treatment. The erythropoietic response to bleeding was quantitated by morphological, gravimetric, and radioiron methods 48 hr after bleeding. At 120 days after ADR, prebleeding base-line cellularity parameters were, in general, similar to those found in untreated age-matched controls. The response to the anemia stress was compared in drug-treated animals and in age-matched untreated controls, and the response deficit was expressed as residual injury (RI). At 120 days, ADR-induced RI was observed to be dose dependent in both femoral marrow and spleen. ADR-induced RI in femoral marrow and spleen were similar at 45 and 120 days, with no significant recovery. Although marrow RI was noted 45 days after 200 mg 1-beta-D-arabinofuranosylcytosine per kg, there was no RI at 120 days. The results indicate that ADR can induce a long-lasting hematopoietic injury which is not obvious from measures of homeostatic cellularity, but which can be expressed after induction of an acute proliferative demand.

In vitro labeling and gold activation autoradiography for determination of labeling index and DNA synthesis times of solid tumors.

In vitro labeling and gold activation autoradiography were used to determine the [3H]thymidine ([3H]TdR)-labeling indices and DNA synthesis times for C3H/He spontaneous mammary tumors. Three variations of the [3H]TdR, [14C]thymidine ([14C]TdR) double-labeling method, together with double-emulsion autoradiography, were used to determine the DNA synthesis times (TS). Tumors labeled totally in vivo (in vivo-in vivo method) and tumors labeled with [3H]TdR in vivo and subsequently labeled with [14C]TdR in vitro showed similar TS values. DNA synthesis times for tumors determined totally in vitro by double labeling (in vitro-in vitro method) were significantly longer than those observed in vivo; however, identical samples subjected to Hypaque-Ficoll gradient separation after double labeling showed TS's similar to those found in vivo. Furthermore, the interval between [3H]TdR and [14C]TdR administration had no effect on TS estimates in vitro. Gold activation autoradiography was used in the present experiments to reduce autoradiographic exposure times. This method, together with in vitro labeling, permits [3H]TdR labeling index and TS determinations after 6-hr and 7-day exposures, respectively.

Antitumor effects of recombinant human interleukin 1 alpha in RIF-1 and Panc02 solid tumors.

The antitumor effects of recombinant human interleukin 1 alpha (IL-1) were determined in RIF-1 and Panc02 murine solid tumors. Acute tumor hemorrhage was observed in both models as early as 3 h after a single 25 micrograms/kg IL-1 treatment and was quantitated by the intratumor accumulation of 59Fe-labeled erythrocytes (RBC). The IL-1-mediated hemorrhagic response was maximal 6-12 h after treatment and greater in Panc02 tumors than in RIF-1 tumors. Hemorrhagic responses to RIF-1 tumors growing in athymic nude mice were similar to those seen for RIF-1 tumors in C3H/HeJ mice. This acute vascular injury was accompanied by progressive edema in tumors but not in skin or muscle. In RIF-1 tumors, the extracell water volume at 12 h after IL-1 (395 microI/g) was nearly twice that in untreated controls (215 microI/g). IL-1 also produced marked reductions in tumor blood flow as early as 1 h after treatment. Maximal blood flow restriction was seen at 6 h after IL-1. Although restricted blood flow was observed in tumors for up to 48 h, IL-1 effects on muscle, liver, and skin blood flow were transient with recovery by 12 h after treatment. IL-1 (up to 0.4 ng/ml for 72 h) was not toxic to RIF-1 tissue culture cells in vitro, but 0.2 ng/ml IL-1, for 20 h, reduced the clonogenicity of RIF-1 cells in primary explant cultures by approximately 50%. In vivo, the clonogenic cellularity of RIF-1 tumors was reduced by 70%, 24 h after a single 25 micrograms/kg treatment. Increased clonogenic cell proliferation was observed at 24 h, and rapid repopulation of the clonogenic cell population was seen by 48 h. Although IL-1 transiently slowed the growth of RIF-1 tumors, no significant regrowth delay was observed. In Panc02 tumors, cell proliferation was also inhibited after IL-1. Recovery, however, was delayed and occurred more slowly than in RIF-1 tumors. Significant growth inhibition and regrowth delay (5 days) was observed in Panc02 tumors after a single IL-1 treatment. The results of these studies show that IL-1 has significant effects on the pathophysiology of both RIF-1 and Panc02 tumors in vivo. Further, our results indicate that these effects may be mediated through the activation of a non T-cell, adherent cell population residing in the tumor at the time of IL-1 treatment.

Potentiation of mitomycin C and porfiromycin antitumor activity in solid tumor models by recombinant human interleukin 1 alpha.

The time- and dose-dependent effects of recombinant human interleukin 1 alpha (IL-1 alpha) on the antitumor activity of mitomycin C (MMC) and porfiromycin (PORF) were studied in RIF-1 and Panc02 solid tumor model systems. IL-1 alpha produced dose-dependent sensitization of clonogenic RIF-1 tumor cells to MMC in vivo. IL-1 alpha chemosensitization was highly schedule dependent, and the most efficacious schedules produced dose-modifying factors of 3.6 and 5.1 for MMC and PORF, respectively. More than additive clonogenic cell kill after IL-1 alpha-chemotherapy combinations reflected increased cellular sensitivity to MMC and PORF. The combinations also produced marked decreases in the yield of viable tumor cells, suggesting that the bioreductive drugs may have also potentiated the microvascular injury and ischemia produced by IL-1 alpha. Dexamethasone inhibited and ketoconazole, an inhibitor of corticosterone biosynthesis, enhanced IL-1 alpha-mediated chemosensitization in these models. IL-1 alpha mediated chemosensitization to MMC, and PORF was also demonstrated by tumor growth inhibition in the RIF-1 model and increased survival of mice in the spontaneously metastasizing Panc02 system. Chemosensitization of bone marrow spleen colony-forming units was not seen. IL-1 alpha (1000 units/ml) had no effect on MMC and PORF cytotoxicity in RIF-1 and PORF cell lines in vitro. The results indicate that the tumor-specific IL-1 alpha-induced pathophysiologies can sensitize solid tumors to agents which are preferentially activated, retained, and cytotoxic to cells under hypoxic conditions. Our results suggest that strategies combining bioreductively activated hypoxic cell cytotoxins and biological agents might offer efficacious alternatives or adjuvants to conventional combination approaches.

Synergistic antitumor activity of cisplatin and interleukin 1 in sensitive and resistant solid tumors.

The antitumor activity of cis-diamminedichloroplatinum(II) (cP) and human recombinant interleukin-1 alpha (IL-1 alpha) was studied in RIF-1 and SC VII solid tumor models and in a cP-resistant subline of RIF-1 designated RIF-R1cP. In RIF-1 tumors, clonogenic cell survival after cP plus IL-1 alpha combinations was highly schedule and IL-1 alpha dose dependent. More than additive clonogenic cell kill was seen when cP was given 6 h before, but not 8 h before or at 2-6 h after IL-1 alpha. Time course studies indicated that maximal clonogenic cell killing was achieved within 4-6 h after the cP plus IL-1 alpha combination, with little or no recovery for up to 24 h. In vivo dose-response studies indicated that cP plus IL-1 alpha combinations induced more clonogenic cell kill than cP alone in all three tumor models, and analysis by the median effect principle indicated highly synergistic antitumor activity. Dexamethasone but not indomethacin inhibited the synergistic interaction. IL-1 alpha had no effect on the cytotoxicity of cP in SCC VII cells in vitro, and neither in vitro hypoxia nor in vivo ischemia, induced by clamping tumor blood supply, significantly affected cP clonogenic cell killing. Increased clonogenic cell killing was seen, however, after removal of the clamp, implicating reperfusion events, such as oxyradical stress, as a potential mechanism for increased cP cytotoxicity in SCC VII solid tumors. The data from our model systems provide a rationale for additional work to define the mechanisms of the synergistic antitumor activity of the cP plus IL-1 alpha combination and indicate that IL-1 alpha might be a useful adjunct to increase the clinical efficacy of cP-containing strategies for both sensitive and cP-resistant cancers.

31P-nuclear magnetic resonance studies of the effect of recombinant human interleukin 1 alpha on the bioenergetics of RIF-1 tumors.

The effect of a single injection of human recombinant interleukin 1 alpha (IL-1 alpha) on s.c. RIF-1 tumors in mice was studied by in vivo 31P nuclear magnetic resonance spectroscopy. Spectra were obtained before and up to 24 h after IL-1 alpha. At 2, 4, 6, and 8 h after IL-1 alpha injection, RIF-1 tumors exhibited a reduction in bioenergetic status compared to untreated controls. The Pi to beta-nucleoside triphosphate and the phosphomonoester to beta-nucleoside triphosphate ratios increased, while the phosphocreatine to Pi and phosphodiester to phosphomonoester ratios decreased. Tumor blood flow, estimated by 86RbCl uptake, decreased within 30 min after IL-1 alpha treatment. Minimum perfusion was detected at 4 h, with recovery between 6 and 12 h after IL-1 alpha treatment. Histological sections of the RIF-1 tumors revealed intravascular congestion by 2 h, extravascular hemorrhage by 4 h, and necrosis by 12 h after treatment with IL-1 alpha. The time course of bioenergetic changes in RIF-1 tumors determined by 31P-NMR spectroscopy was found to parallel the reduction and subsequent recovery of tumor blood flow.

Potentiation of interleukin 1 alpha mediated antitumor effects by ketoconazole.

In the present studies, the regulatory role of adrenal hormones on the antitumor activity of recombinant human interleukin 1 alpha (IL-1 alpha) was investigated. Ketoconazole, a potent but transient inhibitor of adrenal steroid hormone biosynthesis, inhibited IL-1 alpha induced increases in plasma corticosterone. In s.c. RIF-1 tumors (C3H/HeJ mice) ketoconazole potentiated IL-1 alpha induced hemorrhagic necrosis (59Fe labeled RBC uptake) and prolonged intervals of low tumor perfusion (86Rb+ uptake) and attendant depletion of tumor high energy phosphate reserves as determined by in vivo 31P nuclear magnetic resonance spectroscopy. In normal muscle and skin the ketoconazole-IL-1 alpha combination had no effect on RBC content and little or no effect on tissue perfusion. Ketoconazole potentiation of IL-1 alpha induced tumor pathophysiologies was accompanied by time and ketoconazole dose dependent potentiation of RIF-1 tumor clonogenic cell killing. Although ketoconazole at 40 mg/kg and IL-1 alpha at 25 micrograms/kg alone each produced approximately 50% clonogenic cell kill, a combined treatment (IL-1 alpha 1 h after ketoconazole) resulted in surviving fractions of approximately 1.5%. In vitro, ketoconazole and IL-1 alpha induced only additive clonogenic cell kill in primary RIF-1 explant cultures. The effect of elevated plasma corticosterone levels, induced by ketamine-acepromazine anesthesia, on IL-1 alpha responsiveness was also studied in the RIF-1 tumor model. In C3H/HeJ mice, anesthesia increased plasma corticosterone levels within 30 min, abrogated the IL-1 alpha effect on tumor perfusion, and prevented depletion of tumor high energy phosphate metabolite reserves. Our results are consistent with the hypothesis that IL-1 alpha mediated adrenal hormone responses exert a profound negative feedback on IL-1 alpha antitumor activities. Our data also indicate that adrenal steroid hormone biosynthetic pathways could provide a focus for modulation strategies to increase the efficacy of cytokine based therapeutic interventions.

NMR study of in vivo RIF-1 tumors. Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances.

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the 31P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the 31P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The 1H and 13C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.