Lewis M. Schiffer

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.

The cell kinetics of human mammary cancers

The cellular kinetics of human mammary tumors were studied by in vitro methods. These techniques include single 3HTdR labeling to measure the 3HTdR LI, double labeling with 3HTdR and 14CTdR to measure DNA synthesis time, and an estimation of the growth fraction by the PDP index. Calculations of the potential doubling time and cell cycle time were made from these measurements. The 3HTdR LI of primary malignant tumors was greater than that of benign tumors, but only half that of metastatic lesions. There was considerable heterogeneity in the 3HTdI LI of primary tumors, but the DNA synthesis times were relatively invariant. Estimation of the growth fraction by the PDP index also revealed extensive heterogeneity, but the primary tumors were not different from metastases. There appear to be subsets of tumors with high and low proliferative values that correlate with some clinical parameters, such as age and nodal positivity. This material provides a data base for stratification of patients for future protocols and the use of cell kinetics in treatment programs.

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.

Preliminary observations on the correlation of proliferative phenomena with in vivo 31P NMR spectroscopy after tumor chemotherapy.

The pioneering studies of Gene Cronkite, and his associates, on the methodology, theory, and reality of mammalian cell kinetics are well known. During the initial years of study of animal and human hematopoiesis, there was a tacit assumption that, in addition to dissecting the physiology of proliferating systems, we would eventually use cell kinetic techniques for the management of various hematologic and malignant conditions. This has been very slow in coming to fruition which has been disappointing to many scientists and clinicians. The reasons for it are quite clear, however, and

AUTORADIOGRAPHIC DETECTION OF DNA POLYMERASE CONTAINING NUCLEI IN SARCOMA 180 ASCITES CELLS

A technique has been developed allowing the autoradiographic detection of incorporation of 3H‐thymidine‐5′‐triphosphate (3H‐TTP) into nuclear DNA of smears of Sarcoma‐180 (S‐180) mouse ascites tumors under the direction of the cells own nuclear DNA polymerase. Dried smears are dipped into an agar solution, which strips cytoplasm from the nuclei, and are then air dried and incubated with a buffered mixture containing four nucleotide triphosphates (one labeled), Mg++, and Ficoll, with the cells own DNA acting as primer. The incorporation of 3H‐TTP into the nuclei, like the cell free DNA polymerase assay, is largely dependent on the presence of all four nucleotide triphosphates and Mg++ and produces a product which is DNase sensitive and RNase resistant.

Cell kinetic alterations in murine mammary tumors following fasting and refeeding

Abstract In the present study, nutritional deprivation was shown to markedly alter the growth and cell kinetics of two widely different experimental tumors, the T-1699 transplantable mammary tumor (TMT) and the C3H HeJ spontaneous mammary tumor (SMT). In vitro techniques were used to determine the thymidine labeling index ([ 3 H]TdR LI), the tumor growth fraction, by the primer available DNA polymerase assay (PDP), and the DNA synthesis time (T S ) following fasting and refeeding. In both systems, fasting resulted in suppression of cell proliferation and significant tumor regression. Refeeding re-established the prefasting growth rates and cell kinetic profiles. The kinetic response to fasting in the TMT involved a generalized lengthening of the cell cycle (T C ), while the timing and magnitude of the response to refeeding appear to be dose dependent. In contrast, the kinetic data from the SMT during fasting was consistent with the presence of a cell cycle block in G 1 . Removal of this block by refeeding was prompt and was characterized by tumor cell synchronization and recruitment of non-cycling cells.

Tumor cell proliferation and sequential chemotherapy after partial tumor resection in C3H/HeJ mammary tumors

SummaryChanges in tumor cell proliferation and chemosensitivity after partial (up to 3/4) surgical resection were studied in C3H/HeJ mammary tumors, In all studies surgical cytoreduction stimulated tumor cell proliferation in the residual tumor, as evidenced by increases in the3H-TdR labelling index between 24 and 72 hrs after surgery. The time of maximal proliferative activity was directly related to the amount of tumor removed. In spontaneous mammary tumors, proliferative recovery after 1/2 resection could be delayed by giving dexamethasone 24 hrs after resection. The regrowth delay after surgery and various combinations of dexamethasone, vincristine, 5-FU, and cyclophosphamide, was used to assess the significance of the kinetic changes in the residual tumor for the design of adjuvant therapeutic strategies.The results indicated that the chemosensitivity of C3H/HeJ mammary tumors was substantially increased after surgical cytoreduction, and that these post-surgical changes were directly correlated with changes in tumor cell proliferative activity.

The cytokinetic basis for the design of efficacious radiotherapy protocols

Abstract Studies were performed to investigate the perturbing effects of ionizing radiations on the cell kinetics of T1699 transplantable mouse mammary tumors (MMT). Cell kinetic parameters included the 3H-TdR labeling index (LI), the DNA synthesis time (TS) and the primer dependent DNA polymerase labeling index (PDPI), an estimate of tumor growth fraction; they were determined by in vitro methods at various times after acute (400–1200 R) and fractionated (1000 R) X-irradiation. The cell kinetic response after both acute and fractionated treatment was generally similar and could be divided into four phases. (1) The initial phase of the response, occurring within the first 24 hr, was characterized by a decrease in 3H-TdR LI, an increase in PDPI and a concomitant lengthening of the TS. (2) The second phase was characterized by a variable, dose dependent interval of decreased cellular proliferation, as evidenced by a lengthening of TS and decreases in both PDPI and 3H-TdR LI. (3) The third phase of the response, the recovery phase, was characterized by increased cellular proliferation as evidenced by the recovery of TS to normal and increases in both PDPI and 3 H-TdR LI to above control levels, in most cases. (4) The fourth phase of the response was characterized by a reestablishment of normal proliferative patterns. Studies with continuing radiotherapy schedules showed that the most effective schedules for local control and ILS were those in which radiation fractions were given just prior to initiation of observed proliferative recovery. The least effective schedules were those in which radiation fractions coincided with the time of maximal proliferative activity.

The measurement of extracellular water volumes in tissues by gadolinium modification of 1H-NMR spin lattice (T1) relaxation

The present studies were conducted in RIF-1, M5076 and Panc02 murine tumor models to compare extracellular water measurements made by 51Cr-EDTA dilution techniques and a new method which exploits the concentration dependent modification of 1H-NMR proton relaxation by Gadolinium-DTPA-dimethyl glucamine (Gd-DTPA-dimeg) in plasma and tissues. The time dependent changes in T1 modification in tissue and plasma were determined at various intervals after i.v. injection of 0.1 ml of 100 mM Gd-DTPA-dimeg and Gd concentrations determined from standard curves of Gd concentration dependent T1 modification of mouse plasma. Comparison of tissue and plasma Gd concentrations permitted the calculation of Gd-DTPA-dimeg distribution volumes. In unperturbed RIF-1, M5076 and Panc02 tumors, Gd-DTPA-dimeg distribution volumes determined by the T1 modification technique were similar to extracellular water spaces determined by 51Cr-EDTA dilution assays. In mouse liver, the 51Cr-EDTA assay resulted in artifactually high extracellular water space estimates due to internalization of the probe; Gd-DTPA-dimeg distribution volumes determined in liver with both the 153Gd-DTPA-dimeg and by the T1 modification method were approximately 150 ul/gram. The Gd-DTPA-dimeg T1 modification method also provided good approximations of changes in extracellular water volumes in RIF-1 tumors after dexamethasone and cyclophosphamide treatments. These results indicate that Gd-DTPA-dimeg modification of proton relaxation may be extremely useful for monitoring changes in tumor water dynamics during cancer therapy.

Human colonic tumor cell kinetics: potential for therapy.

Using new in vitro techniques developed at the Cancer Research Unit, cell kinetic measurements were obtained in primary and metastatic human colonic tumors, polyps and normal bowel that did not require in vivo 3HTdR and required only single samples of tissue. These techniques included the measurement of the number of cells in DNA synthesis (LI), an estimate of the DNA synthesis time (T8) and the growth fraction of tissues by means of the primer‐available DNA‐dependent DNA polymerase assay (PDP). From these data, the potential doubling time and the cell cycle time (Tc) of the tumors were calculated. Early‐preliminary data on human colonic specimens presented in Tables 1 and 2 indicate that there is an increase in LI from the low polyps to higher adeno‐carcinomas. There is little difference between primary and metastatic tumor cell kinetics. Growth fraction estimates (PDP) of the various colonic tissue types are also not significantly different and, except for villous adenomas, DNA synthesis times are constant. The median 3HTdR labeling indices of 7% primary adenocarcinomas include a number of samples (approximately 20% of all samples) with high labeling indices (in the 10‐20% range). These high labeling tumors may be those that show objective response to S‐phase active drugs, e.g., 5‐FU.