Erythropoietic and reticuloendothelial function in bone marrow in dogs.

Erythropoietic and reticuloendothelial functions in bone marrow were found to be identically distributed between various bones and within individual bones in the dog.

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.

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.

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.

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.

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.

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.

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.

Estimation of tumor growth fraction in murine tumors by the primer-available DNA-dependent DNA polymerase assay.

The tumor growth fraction measured by the percentage labeled mitoses method has been determined in transplantable solid and ascites murine tumors, the latter being measured at different times after transplantation. These values were compared to an in vitro, autoradiographic assay that determines the fraction of cells in a given population (primer-available DNA-dependent DNA polymerase index) that have both nuclear DNA-dependent DNA polymerase and DNA capable of acting as primer-template. It appears that almost all cells with a short G1 phase duration (less than 19 hr) that are within the proliferative cycle are primer-available DNA-dependent DNA polymerase positive. The results of the comparison indicate that the primer-available DNA-dependent DNA polymerase index estimation of growth fraction is very nearly identical to the growth fraction measured by the percentage labeled mitoses method.

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.

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.

1H-NMR relaxation times and water compartmentalization in experimental tumor models.

The present studies were conducted with RIF-1, M5076 and Panc02 subcutaneous tumor models to assess the relationship between tissue-free water compartmentalization and observed tissue T1 and T2 changes at 10 MHz. Observed T1 was shown to correlate directly with total extracellular water and interstitial water volumes. T1 and T2 were also inversely related to intracellular water volumes. T1 and T2 decreases after dexamethasone treatment were, however, most closely correlated with changes in tumor extracellular water and not changes in cell or total water volumes. Studies to assess Gd-DTPA-dimeg dose dependent T1 and T2 modification in model serum protein solutions indicated that although the Gd concentration that reduced T2 by 50% was about 2.5 fold greater than that required to reduce T1 equally, the of the concentration dependent T1 and T2 modifications were similar. In studies with tumor models, the injected dose of Gd-DTPA-dimeg that reduced T1 by 50% was inversely correlated with tumor extracellular water volumes. The slopes for dose dependent T1 modification in all tumors were similar and similar to that observed for model protein solutions. Gd-DTPA-dimeg had a different effect on observed T2 values for the 3 tumor models. Exponential slopes were about twice that observed for T2 modification of serum protein solutions, and Gd-DTPA-dimeg doses that reduced observed tumor T2 ranged from 9 to 50 times that necessary to similarly reduce T1. The results from these studies indicate that the observed T1, for these tumors, was dominated by relaxation of water protons in interstitial water but that the observed T2 was most strongly influenced by proton relaxation in water compartments that were unavailable to the Gd labeled probe.

Fluorine nuclear magnetic resonance: calibration and system optimization.

Fluorine-19 magnetic resonance imaging (MRI) offers advantages for imaging organs and tissues. 19F is readily synthesized into a variety of compounds and offers the potential for in-vivo imaging as a complement to hydrogen MRI. The purpose of this work was to determine the minimum detection sensitivity for a fluorinated compound (CF3-CO2H) as a function of pulse sequence, interpulse times (TE, TI, and TR), gradient values and the number of data averages. CF3-CO2H was chosen because it has a single spectral line and exhibits a minimal frequency shift under the experimental conditions used for this experiment. A resistance MR scanner operating at a resonance frequency of 6.255 MHz was used for imaging both fluorine (.156 T) and hydrogen (.147 T). Critical factors determining the minimum detection sensitivity included system signal-to-noise ratio (S/N), acquisition time, relaxation times (T1, T2), and sample volume. Samples were measured over the range of 0.05 M to 20.0 M and showed a linear relationship between signal strength and concentration. The minimum detection sensitivity was 0.1 M. Use of higher static fields and optimized coils as well as improved system signal-to-noise ratios will improve detection sensitivity. We conclude that imaging of fluorine on low-field system is feasible, although it is necessary to optimize many parameters to maximize detection sensitivity.