Radiation induction of drug resistance in RIF-1 tumors and tumor cells

The RIF-1 tumor cell line contains a small number of cells (1-20 per 10(6) cells) that are resistant to various single antineoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), and adriamycin (ADR). For 5FU the frequency of drug resistance is lower for tumor-derived cells than for cells from cell culture; for MTX the reverse is true, and for ADR there is no difference. In vitro irradiation at 5 Gy significantly increased the frequency of drug-resistant cells for 5FU, MTX, and ADR. In vivo irradiation at 3 Gy significantly increased the frequency of drug-resistant cells for 5FU and MTX, but not for ADR. The absolute risk for in vitro induction of MTX, 5FU, and ADR resistance, and for in vivo induction of 5FU resistance, was 1-3 per 10(6) cells per Gy; but the absolute risk for in vivo induction of MTX resistance was 54 per 10(6) cells per Gy. The frequency of drug-resistant cells among individual untreated tumors was highly variable; among individual irradiated tumors the frequency of drug-resistant cells was significantly less variable. These studies provide supporting data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be due to radiation-induced drug resistance.     

Blood flow dynamics after photodynamic therapy with verteporfin in the RIF-1 tumor

In the present study, the effects of photodynamic therapy (PDT) with verteporfin on tumor blood flow and tumor regrowth were compared as verteporfin distributed in different compartments within the RIF-1 tumor. Tissue distribution of verteporfin was examined by fluorescence microscopy, and blood flow measurements were taken with a laser Doppler system. It was found that, at 15 min after drug administration, when verteporfin was mainly confined within the vasculature, PDT induced a complete arrest of blood flow by 6 h after treatment. PDT treatment at a longer drug-light interval (3 h), which allowed the drug to diffuse to the tumor interstitium, caused significantly less flow decrease, only to 50% of the initial flow in 6 h. A histological study and Hoechst 33342 staining of functional tumor vasculature confirmed the primary vascular damage and the decrease in tumor perfusion. The regrowth rate of tumors treated with 15-min interval PDT was 64% of that of the control group. However, when tumors were treated with 3-h interval PDT, the regrowth rate was not significantly different from that of the control, indicating that only the 15-min interval PDT caused serious damage to the tumor vascular bed. These results support the hypothesis that temporal pharmacokinetic changes in the distribution of the photosensitizer between the tumor parenchyma and blood vessels can significantly alter the tumor target of PDT.     

RIF-1 tumor treatment in anesthetized mice with minimal effects on blood flow and hypoxia

The injectable anesthetic etomidate and a clip that facilitates hyperthermia by water bath immersion (the "Gibbs clip") were evaluated for their suitability with subcutaneous flank RIF-1 tumors in C3H/HeJ mice. For tumors between 100 and 250 mg (mean, 160 mg), etomidate at 40 mg kg-1 ip did not significantly increase the radiobiologic hypoxic fraction (RHF); as calculated from an in vitro assay after treatment in vivo the RHF increased from 0.06 (95% C.I.:0.03-0.13) to 0.08 (0.04-0.16). In contrast, for larger tumors (270-650 mg; mean, 400 mg) etomidate increased the RHF from 0.08 (0.04-0.17) to 0.28 (0.14-0.60). Holding 250-mg-or-less tumors 3-mm laterally away from the flank in an X-ray jig did not significantly reduce tumor blood flow as inferred from the clearance rates of Xe, but the RHF of 0.15 (0.08-0.26) was significantly (P less than 0.05) greater than the RHF in unanesthetized mice, although not the RHF in anesthetized mice. The Gibbs clip, which folded skin around a tumor to enhance thermal conduction from a water bath, did not impair the increase in tumor blood flow in response to the cardiovascular arousal associated with exposure to a hyperthermic stimulus. Intratumor temperature was within 0.25 degrees C of bath temperature 3 min after the tumor and clip were immersed, but only when rectal temperatures were at 37 degrees C or above; tumor blood flow increased intratumor temperature gradients by 0.10 degrees C for each 1.5 degrees C that the body temperature was below 37 degrees C.     

Photoactivation of lysosomally sequestered sunitinib after angiostatic treatment causes vascular occlusion and enhances tumor growth inhibition

The angiogenesis inhibitor sunitinib is a tyrosine kinase inhibitor that acts mainly on the VEGF and PDGF pathways. We have previously shown that sunitinib is sequestered in the lysosomes of exposed tumor and endothelial cells. This phenomenon is part of the drug-induced resistance observed in the clinic. Here, we demonstrate that when exposed to light, sequestered sunitinib causes immediate destruction of the lysosomes, resulting in the release of sunitinib and cell death. We hypothesized that this photoactivation of sunitinib could be used as a vaso-occlusive vascular-targeting approach to treating cancer. Spectral properties of sunitinib and its lysosomal accumulation were measured in vitro. The human A2780 ovarian carcinoma transplanted onto the chicken chorioallantoic membrane (CAM) and the Colo-26 colorectal carcinoma model in Balb/c mice were used to test the effects of administrating sunitinib and subsequently exposing tumor tissue to light. Tumors were subsequently resected and subject to immunohistochemical analysis. In A2780 ovarian carcinoma tumors, treatment with sunitinib+light resulted in immediate specific angio-occlusion, leading to a necrotic tumor mass 24 h after treatment. Tumor growth was inhibited by 70% as compared with the control group (**P<0.0001). Similar observations were made in the Colo-26 colorectal carcinoma, where light exposure of the sunitinib-treated mice inhibited tumor growth by 50% as compared with the control and by 25% as compared with sunitinib-only-treated tumors (N≥4; P=0.0002). Histology revealed that photoactivation of sunitinib resulted in a change in tumor vessel architecture. The current results suggest that the spectral properties of sunitinib can be exploited for application against certain cancer indications.Angiogenesis inhibitors are currently firmly implemented in the clinical management of cancer. For example, sunitinib has been approved for the treatment of advanced renal cell carcinoma (RCC),¹ gastrointestinal stromal tumors (GIST)² and pancreatic neuroendocrine tumors.³ Studies assessing its activity against other tumor types are currently underway.⁴ Sunitinib was developed as an angiogenesis inhibitor⁵ and revolutionized the management of advanced RCC and GIST. Its mode of action is based on the suppression of the tyrosine kinase activity of several growth factor receptors, mainly VEGFR2 and PDGFR (alpha and beta).⁶ It has been previously shown that sunitinib is sequestered by tumor cells in their lysosomal compartments.⁷ This sequestration, which limits the exposure of other parts of the cell to sunitinib, is part of a drug-induced resistance mechanism that has also been clinically observed.⁷ The sequestration and accumulation of sunitinib in the lysosomes is similar to a phenomenon that has been described for certain other chemotherapeutics^{8, 9} or photosensitizing compounds,¹⁰ and depends, at least to some extent, on the hydrophobic and weak basic features of the molecule. We discovered a particular characteristic of sunitinib based on its optical properties, which may be helpful in the treatment of certain cancers. On the basis of the spectral features of sunitinib, we hypothesized that the drug may have photosensitizer-like properties.¹¹ If so, exposure to light of an appropriate wavelength could lead to the disruption of the lysosomal membrane, the release of sunitinib and re-exposure of the cell to this molecule. This could lead to further cell damage and eventually cell death. In this study, we show that the exposure of sunitinib-treated cells to light of an appropriate wavelength excites the molecule, which leads to the generation of reactive oxygen species (ROS). We show in two tumor models, that is, human ovarian carcinoma (A2780) xenografted on the chorioallantoic membrane of the chicken embryo (chicken chorioallantoic membrane (CAM)) and in colorectal carcinoma in Balb/c mice, that treatment with sunitinib and its subsequent photoexcitation leads to significant occlusion of the vasculature and inhibition of tumor growth. Our results indicate that additional antitumor activity can be obtained after the normal use of sunitinib through its use as a photosensitizer and the application of light. The combination of classical sunitinib-induced angiostasis with the re-exposure of tumor cells to sunitinib after the destruction of lysosomes and photodynamic vessel obstruction can lead to a strategy that may be applicable in patients.     

Radiosensitivity and thermosensitization of thermotolerant Chinese hamster cells and RIF-1 tumors

CHO cells subline HA-1 were made thermotolerant by a priming heat treatment (43 degrees C, 30 min). Later, 4, 16, or 24 hr, they were either irradiated or heated (43 degrees C, 30 min) and irradiated. Thermotolerance had no effect on the radiation sensitivity of the cells as measured by the D0 value of the clonogenic survival curve. However, the N value of the curve (width of shoulder) showed a significant increase at 24 hr, indicating an increased capacity to accumulate sublethal damage. This indicates that the fractionation schedule 43 degrees C, 30 min + 37 degrees C, 24 hr + 43 degrees C, 30 min + X ray required approximately 100 rad more radiation than 43 degrees C, 30 min + X ray to reduce survival to the same level. The same priming treatment was given to RIF-1 tumors growing in C3H mice. Later, 24 hr, when the tumors were either irradiated or heated (43 degrees C, 30 min) and irradiated, it was found that thermotolerance had no effect on the radiosensitivity of the cells as measured by in vitro assay. However, thermal radiosensitization was not apparent 24 hr after the priming treatment.     

Effects of hydralazine on the blood flow in RIF-1 tumors and normal tissues of mice

Hydralazine is a peripheral vasodilator used as an antihypertensive agent. Hydralazine has been reported to potentiate tumor damage by hyperthermia as well as by hypoxic-cell-specific drugs through the reduction of tumor blood flow and pO2. In the present study, we investigated the changes in blood perfusion caused by hydralazine in S.C. RIF-1 tumors and normal tissues in C3H mice using the 86Rb uptake technique and laser Doppler flowmetry. The tumor blood flow was decreased significantly by an intravenous administration of 0.5-10.0 mg/kg hydralazine, as determined by both uptake of 86Rb and laser Doppler flowmetry. The tumor pO2 was also decreased significantly by the injection of hydralazine. On the other hand, the uptake of 86Rb was increased significantly in the skin and muscle by hydralazine. The changes seen in the skin and muscle after injection of hydralazine as assessed by laser Doppler flowmetry were similar to those assessed by uptake of 86Rb, indicating a significant increase in blood circulation in these tissues. Uptake of 86Rb remained unchanged in the kidney and decreased in the liver and spleen in the presence of hydralazine in a dose-dependent manner at 0.5-10.0 mg/kg. The decline in uptake of 86Rb in normal tissues strongly suggests that hydralazine decreases the blood flow in these normal tissues. Thus the recent proposal to use hydralazine to increase the antitumor activity of heat or certain drugs needs to be reexamined.     

Comparison of hormone responses following light resistance exercise with partial vascular occlusion and moderately difficult resistance exercise without occlusion.

Previous studies of contracting muscle with low loading and partial vascular occlusion demonstrated hypertrophy and strength adaptations similar to and exceeding those observed with traditional moderate to high resistance (Shinohara M, Kouzaki M, Yoshihisa T, and Fukunaga T. Eur J Physiol 77: 189-191, 1998; Takarada Y, Takazawa H, Sato Y, Takebayashi S, Tanaka Y, and Ishii N. J Appl Physiol 88: 2097-2106, 2000; Takarada Y, Sato Y, and Ishii N. Eur J Physiol 86: 308-314, 2002). The purpose of the study was to determine the anabolic and catabolic hormone responses to light resistance exercise combined with partial vascular occlusion. Three experimental conditions of light resistance with partial occlusion (LRO), moderate resistance with no occlusion (MR), and partial occlusion without exercise (OO) were performed by eight healthy subjects [mean 21 yr (SD 1.8)]. Three sets of single-arm biceps curls and single-leg calf presses were completed to failure with 1-min interset rest periods. Workloads of 30 and 70% one repetition maximum for each exercise were lifted for the LRO and MR trials, respectively. Blood samples were taken preexercise, postexercise, and 15 min postexercise for each experimental condition. Lactate increased significantly in the LRO and MR trials and was not significantly different from each other at any time point. Growth hormone (GH) increased significantly by fourfold from pre- to postexercise in the LRO session but did not change significantly during this time period in the MR and OO trials (8.3 +/- 2.3 vs. 2.1 +/- 1.2 and 2.6 +/- 0.94 microg/l; respectively, P < 0.05). There were no changes in resting total testosterone [T; mean 15.7 +/- 1.6 (SE) nmol/l], free testosterone (FT; 54.1 +/- 4.5 pmol/l), or cortisol (267.6 +/- 22 nmol/l) across all trials and times. In conclusion, with similar lactate responses, light exercise combined with partial vascular occlusion elicits a greater GH response than moderate exercise without occlusion but does not affect T, FT, or cortisol.     

Comparison of the protective effects of three phosphorothioate radioprotectors in the RIF-1 tumor

Three aminoalkyl phosphorothioates, WR-2721, WR-3689, and WR-77913, were compared as radioprotectors of RIF-1 tumors irradiated in vivo and assayed for cell survival in vitro. The protector doses were 50% of the acute drug LD50. The radiation dose modifying factors for the three drugs were nearly equal, ranging from 1.5 to 1.7 at surviving fractions of 0.1 and 0.05. Using biodistribution data obtained with 35S labeled drugs, the uptake in tumors was calculated as micromoles drug per gram of tumor. On this basis, tumor levels of WR-77913 were 4.5-fold those of WR-2721, and WR-3689 uptake was 2.7-fold greater than uptake of WR-2721. Thus, on a molar basis, WR-2721 appears to be the most effective protector, but all three phosphorothioates protect this tumor moderately well. In diffusible substance autoradiographs of 3H WR-3689 labeled tumors, label was generally distributed over cells with no evidence of preferential localization over nuclei.     

Rapid increase in plasma growth hormone after low-intensity resistance exercise with vascular occlusion.

Hormonal and inflammatory responses to low-intensity resistance exercise with vascular occlusion were studied. Subjects (n = 6) performed bilateral leg extension exercise in the seated position, with the proximal end of their thigh compressed at 214 +/- 7.7 (SE) mmHg throughout the session of exercise by means of a pressure tourniquet. Mean intensity and quantity of the exercise were 20% of 1 repetition maximum and 14 repetitions x 5 sets, respectively. In each set, the subjects repeated the movement until exhaustion. Plasma concentrations of growth hormone (GH), norepinephrine (NE), lacate (La), lipid peroxide (LP), interleukin-6 (IL-6), and activity of creatine phosphokinase (CPK) were measured before and after the exercise was finished and the tourniquet was released. Concentrations of GH, NE, and La consistently showed marked, transient increases after the exercise with occlusion, whereas they did not change a great deal after the exercise without occlusion (control) done at the same intensity and quantity. Notably, concentration of GH reached a level approximately 290 times as high as that of the resting level 15 min after the exercise. IL-6 concentration showed a much more gradual increase and was maintained at a slightly higher level than in the control even 24 h after exercise. Concentrations of LP and CPK showed no significant change. The results suggest that extremely light resistance exercise combined with occlusion greatly stimulates the secretion of GH through regional accumulation of metabolites without considerable tissue damage.     

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo and in vitro. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose. The D0 of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.     

Venous occlusion pressure and vascular permeability in the dog lung after air embolization

Pulmonary edema has frequently been associated with air embolization of the lung. In the present study the hemodynamic effects of air emboli (AE) were studied in the isolated mechanically ventilated canine right lower lung lobe (RLL), pump perfused at a constant blood flow. Air was infused via the pulmonary artery (n = 7) at 0.6 ml/min until pulmonary arterial pressure (Pa) rose 250%. While Pa rose from 12.4 +/- 0.6 to 44.6 +/- 2.0 (SE) cmH2O (P less than 0.05), venous occlusion pressure remained constant (7.0 +/- 0.5 to 6.8 +/- 0.6 cmH2O; P greater than 0.05). Lobar vascular resistance (RT) increased from 2.8 +/- 0.3 to 12.1 +/- 0.2 Torr.ml-1.min.10(-2) (P less than 0.05), whereas the venous occlusion technique used to determine the segmental distribution of vascular resistance indicated the increase in RT was confined to vessels upstream to the veins. Control lobes (n = 7) administered saline at a similar rate showed no significant hemodynamic changes. As an index of microvascular injury the pulmonary filtration coefficient (Kf) was obtained by sequential elevations of lobar vascular pressures. The Kf was 0.11 +/- 0.01 and 0.07 +/- 0.01 ml.min-1.Torr-1.100 g RLL-1 in AE and control lobes, respectively (P less than 0.05). Despite a higher Kf in AE lobes, total lobe weight gains did not differ and airway fluid was not seen in the AE group. Although air embolization caused an increase in upstream resistance and vascular permeability, venous occlusion pressure did not increase, and marked edema did not occur.     

The effects of Efaproxyn (efaproxiral) on subcutaneous RIF-1 tumor oxygenation and enhancement of radiotherapy-mediated inhibition of tumor growth in mice

Efaproxiral, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity, facilitating oxygen release from hemoglobin, which is likely to increase tissue pO(2). The purpose of this study was to determine the effect of efaproxiral on tumor oxygenation and growth inhibition of RIF-1 tumors that received X radiation (4 Gy) plus oxygen breathing compared to radiation plus oxygen plus efaproxiral daily for 5 days. Two lithium phthalocyanine (LiPc) deposits were implanted in RIF-1 tumors in C3H mice for tumor pO(2) measurements using EPR oximetry. Efaproxiral significantly increased tumor oxygenation by 8.4 to 43.4 mmHg within 5 days, with maximum increases at 22-31 min after treatment. Oxygen breathing alone did not affect tumor pO(2). Radiation plus oxygen plus efaproxiral produced tumor growth inhibition throughout the treatment duration, and inhibition was significantly different from radiation plus oxygen from day 3 to day 5. The results of this study provide unambiguous quantitative information on the effectiveness of efaproxiral to consistently and reproducibly increase tumor oxygenation over the course of 5 days of treatment, modeling the clinical use of efaproxiral. Also, based on the tumor growth inhibition, the study shows the efaproxiral-enhanced tumor oxygenation was radiobiologically significant. This is the first study to demonstrate the ability of efaproxiral to increase tumor oxygenation and to increase the tumor growth inhibition of radiotherapy over 5 days of treatment.     

Primary human lymphocytes transduced with NY-ESO-1 antigen-specific TCR genes recognize and kill diverse human tumor cell lines

cDNAs encoding TCR alpha- and beta-chains specific for HLA-A2-restricted cancer-testis Ag NY-ESO-1 were cloned using a 5'RACE method from RNA isolated from a CTL generated by in vitro stimulation of PBMC with modified NY-ESO-1-specific peptide (p157-165, 9V). Functionality of the cloned TCR was confirmed by RNA electroporation of primary PBL. cDNA for these alpha- and beta-chains were used to construct a murine stem cell virus-based retroviral vector, and high titer packaging cell lines were generated. Gene transfer efficiency in primary T lymphocytes of up to 60% was obtained without selection using a method of precoating retroviral vectors onto culture plates. Both CD4(+) and CD8(+) T cells could be transduced at the same efficiency. High avidity Ag recognition was demonstrated by coculture of transduced lymphocytes with target cells pulsed with low levels of peptide (<20 pM). TCR-transduced CD4 T cells, when cocultured with NY-ESO-1 peptide pulsed T2 cells, could produce IFN-gamma, GM-CSF, IL-4, and IL-10, suggesting CD8-independent, HLA-A2-restricted TCR activation. The transduced lymphocytes could efficiently recognize and kill HLA-A2- and NY-ESO-1-positive melanoma cell lines in a 4-h (51)Cr release assay. Finally, transduced T cells could efficiently recognize NY-ESO-1-positive nonmelanoma tumor cell lines. These results strongly support the idea that redirection of normal T cell specificity by TCR gene transfer can have potential applications in tumor adoptive immunotherapy.     

Polyamine modification in human breast tumors after short treatment with tamoxifen

The authors measured estrogen receptors, and polyamine (putrescine, spermidine and spermine) in breast tumors from patients (n = 23) who had received tamoxifen for a few days (5-10) before surgery. Women undergoing mastectomy without any preoperatory treatment were selected as the control group (n = 44). As already reported about in vitro experiments, the treatment resulted in a significant lowering of the spermidine to spermine ratio. Such a modification was larger in the ER positive tumors then in the ER negative ones and it seems to be related to the regression process of the drug-responsive tumors. On the basis of the experimental data the authors suggest the development of a in vivo tamoxifen-sensitivity test.     

Conceptus development after vascular occlusion of the middle uterine artery in the pig

The middle uterine artery of gilts was occluded unilaterally or bilaterally from Days 25 to 70 after mating. The results showed that vascular occlusion of one (N = 7) or both (N = 6) middle uterine arteries during mid-pregnancy markedly reduced, compared with sham-operated controls (N = 7), development of the conceptuses and decreased peripheral oestrogen (oestrone + oestradiol-17 beta) concentrations in maternal blood.     

Defining the pHi-hyperthermia sensitivity relationship for the RIF-1 tumor in vivo: a 31P MR spectroscopy study

This study quantifies the enhancement of the therapeutic efficacy of hyperthermia resulting from an acutely acidified and accurately monitored intracellular pH (pHi) in a mouse tumor model in vivo. Metabolic manipulation of the physiology of RIF-1 tumor (subcutaneous, on the hind flanks of female C3H/HeJ mice) achieved by i.p. bolus injection of glucose (glycolytic tumor acidification) or 3-O-methylglucose (non-glycolytic tumor acidification) was monitored by 31P magnetic resonance (31P MR) prior to, during and up to 1 h after localized hyperthermia. The pre-hyperthermia 31P MR-observable metabolic parameter that correlates most strongly with thermal sensitivity is pHi. Thermal sensitivity increases linearly with decreasing pHi regardless of the mechanism (glycolytic or non-glycolytic) of metabolic manipulation. The quantitative relationship is described by log10(SF)/EQ43=0.0079 pHi,preHT -0.0606 (R=0.63, P<0.0001), where EQ43 is the thermal heat dose delivered to the tumor (in units of equivalent minutes at 42.5 degrees C), pHi,preHT is the intracellular pH immediately prior to hyperthermia, and SF is the surviving fraction. The therapeutic enhancement is not as dramatic as expected based upon previously reported in vitro studies but is generally consistent with other in vivo studies. The method still represents a viable strategy for enhancing the therapeutic efficacy of hyperthermia, especially when used in combination with other therapeutic modalities.     

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 vivo and in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.