Biomedical subjects

S P Tomasovic

Publications and source records attributed to S P Tomasovic.

At least 19 recordsLinked to original sources

PubMed2001

Characteristics of human dendritic cells generated in a microgravity analog culture system.

Generation of an effective immune response requires that antigens be processed and presented to T lymphocytes by antigen-presenting cells, the most efficient of which are dendritic cells (DC). Because of their influence on both the innate and the acquired arms of immunity, a defect in DC would be expected to result in a broad impairment of immune function, not unlike that observed in astronauts during or after space flight. In the study reported here, we investigated whether DC generation and function are altered in a culture environment that models microgravity, i.e., the rotary-cell culture system (RCCS). We observed that RCCS supported the generation of DC identified by morphology, phenotype (HLA-DR+ and lacking lineage-associated markers), and function (high allostimulatory activity). However, the yield of DC from RCCS was significantly lower than that from static cultures. RCCS-generated DC were less able to phagocytose Aspergillus fumigatus conidia and expressed a lower density of surface HLA-DR. The proportion of DC expressing CD80 was also significantly reduced in RCCS compared to static cultures. When exposed to fungal antigens, RCCS-generated DC produced lower levels of interleukin-12 and failed to upregulate some costimulatory/adhesion molecules involved in antigen presentation. These data suggest that DC generation, and some functions needed to mount an effective immune response to pathogens, may be disturbed in the microgravity environment of space.

Antigens, CD34↗

PubMed1996

Murine cells transfected with human Hsp27 cDNA resist TNF-induced cytotoxicity.

Hyperthermia sensitizes tumor cells to killing by tumor necrosis factor-alpha (TNF). Sensitization is greater in cells exposed to TNF before heating begins than with the reverse sequence, and heat-shock proteins (hsp) have been suggested to protect cells from TNF cytotoxicity. Here we examined the role of Hsp27 in TNF resistance. Murine L929 cells were stably transfected with the vector pRc/CMV constitutively to express an inserted human hsp27 complementary DNA (cDNA) sequence. Parental cells produced no detectable murine homolog to human hsp27. Hsp27-sense clones expressed hsp27 messenger RNA (mRNA) and protein at 37 degrees C. Cells transfected with the cDNA in the anti-sense orientation produced anti-sense mRNA but no protein, and cells transfected with the vector alone produced neither product. Expression of hsp27 conferred significant resistance to TNF cytotoxicity in both neutral red cytotoxicity and clonogenic survival assays. Vector along and hsp27 anti-sense transfectants had a TNF response similar to that of parental L929 cells. Kinetic studies in L929 cells showed that hsp27-expressing clones exhibited resistance relative to parental cells beginning 6 h after TNF exposure, and this differential response increased by 12 and 24 h. Addition of actinomycin D to the TNF cytotoxicity assays accelerated the cytotoxicity development in parental and transfected cells, but the hsp27-sense clones were still more resistant. Hsp27-sense clones of L929 cells were also resistant to oxidative stress induced by menadione and released less arachidonic acid in response to TNF induction. These results show that hsp27 can negatively regulate the TNF cytotoxic mechanism.

Animals↗

PubMed1995

Tetramodality treatment of human melanoma in vitro.

We evaluated the in vitro cytotoxic effects of combined human tumour necrosis factor alpha (TNF), human interferon gamma (IFN-gamma), melphalan (L-PAM) and hyperthermia (HTX) on human melanoma cell lines using the crystal violet assay. HTX (40 degrees C, 1 h) alone had no effect. The responses of the cell lines to TNF were in the rank order of 939 cells > 987 > 284 > C8161 > 852 > A2058 approximately 0, and all displayed shallow dose-response curves; no significant thermal enhancement of TNF cytotoxicity was apparent with this heat dose. All cell lines were sensitive to L-PAM, with 284 cells being the most sensitive; HTX caused only slightly increased sensitization to L-PAM. The combination of TNF and L-PAM resulted in generally subadditive or additive cytotoxicity, with or without HTX. The response to IFN-gamma alone was heterogeneous; the 939, 284 and 852 cell lines were sensitive to a dose as low as 20 ng/ml, whereas the 987 line was resistant to 2.0 micrograms/ml, even with HTX. IFN-gamma enhanced the response to TNF only of the TNF-resistant A2058 cell line, but there was no enhancement of the response to L-PAM for any line. Thus, this tetramodality combination achieved generally subadditive or additive cytoxicity in vitro.

Cell Survival↗

PubMed1995

Clonogenic survival studies of human colon tumor cell lines in vitro: combined hyperthermia, 5-fluorouracil/leucovorin, carboplatin and tumor necrosis factor.

The responses of DLD-1 and HCT-15 human colon adenocarcinoma cells to hyperthermia, 5-fluorouracil (5-FU)/leucovorin, carboplatin and tumor necrosis factor-alpha, singly and in multiple combinations, were evaluated in clonogenic assays. The combination of hyperthermia with the lower dose combination resulted in a survival fraction of about 0.005 to 0.001 for both cell types, whereas estimated additive interactions alone would have resulted in a survival fraction of about 0.5 (DLD-1) or 0.05 (HCT-15). A survival fraction of 0.00001 or greater was observed when the higher dose levels were combined with hyperthermia, whereas additive interactions alone would have achieved a decrease of only 0.001 or 0.0001 in the surviving fraction. The combination of the three other modalities at either dose level under conditions of hyperthermia or normothermia achieved statistically significant apparently supra-additive losses of clonogenicity in HCT-15 cells; similar results were obtained with the lower dose level in DLD-1 cells. Our results suggest that human colon tumor cells are markedly sensitive to this combination of modalities when used at clinically achievable dose levels.

Carboplatin↗

PubMed1994

Hyperthermia enhances the cytotoxicity of National Institutes of Health 3T3 cells transfected with a noncleavable transmembrane pro-tumor necrosis factor deletion mutant.

Hyperthermia has been shown to potentiate the cytotoxicity of exogenously added tumor necrosis factor (TNF) against tumor cell targets. The mechanism for that interaction is not known, but among the possibilities are that heat enhances internalization of ligand-bound TNF or enhances processing of internalized TNF. In this study, we found that NIH 3T3 cells transfected with an expression vector containing the full-length human pro-TNF secreted TNF and that hyperthermic treatment of chromium-labeled L929 target cells at 43 degrees C for 1 h potentiated the cytotoxicity of these transfectants against the L929 cells in clonogenic survival and chromium-release assays. On the other hand, transfectants expressing a transmembrane, nonsecretable pro-TNF mutant that kills L929 cells by cell-to-cell contact without internalization also exhibited enhanced cytotoxicity against heated L929 cell targets. Thus, potentiation of the cytotoxicity of TNF by hyperthermia is not strictly dependent on enhanced internalization of ligand-bound TNF or enhanced processing of internalized TNF.

3T3 Cells↗

PubMed1992

Enhanced sensitivity of human colon tumor cell lines in vitro in response to thermochemoimmunotherapy.

We have investigated the cytotoxic responses in vitro of three human colon tumor cell lines with epithelial-like morphology, DLD-1, HCT-15, and HT-29, to thermochemoimmunotherapy with hyperthermia (42 degrees C for 2 h), carboplatin, and recombinant human tumor necrosis factor (TNF). Dose ranges of carboplatin and recombinant human TNF were administered essentially simultaneously and were followed 1 h later by hyperthermia. A two-tiered approach was used to evaluate cytotoxicity. In the first tier, a 5-day microcytotoxicity assay using vital dye staining was done; the effect on surviving fraction of simultaneously varying carboplatin and recombinant human TNF doses was evaluated by response surface methodology. From this analysis doses were selected for use in the second-tier clonogenic survival assays. A similar treatment protocol was used in clonogenic assays. Both assays revealed significant interline treatment response heterogeneity. Only the HCT-15 cells were sensitive to TNF alone; carboplatin activity against all three tumor cell lines was enhanced by TNF. Hyperthermia had minimal effect as a sole agent but enhanced the effects of carboplatin and TNF in DLD-1 and HCT-15 cells. Triple modality treatment resulted in 3-4-log decreased survival and could reduce cytotoxic resistance expressed against single- or dual-modality treatments by some of these cells.

Adenocarcinoma↗

PubMed1992

Increased therapeutic efficacy induced by tumor necrosis factor alpha combined with platinum complexes and whole-body hyperthermia in rats.

This study examined the effect of a trimodality therapy of the combination of recombinant human tumor necrosis factor alpha (TNF), whole-body hypertheria (WBH), and cis-diamminedichloroplatinum(II) (CDDP) or cis-diammine-1,1-cyclobutane dicarboxylate platinum(II) (CBDCA) on a fibrosarcoma and normal tissue in F344 rats. TNF (1 x 10(5) units/kg) increased the antitumor effect of both CDDP (1.5 mg/kg) + WBH (2 h at 41.5 degrees C) and CBDCA (30 mg/kg) + WBH. Tumor growth delay, which was 1.9 days for CDDP + WBH and 2.7 days for CBDCA + WBH (P less than 0.01 compared to control), was significantly increased to 2.9 days with TNF + CDDP + WBH and 5.4 days with TNF + CBDCA + WBH (P less than 0.05). WBH, TNF, CDDP or CBDCA alone, TNF + CDDP, TNF + CBDCA, or TNF + WBH had no significant effect on tumor growth. In contrast, administration of TNF did not enhance the CDDP- or CBDCA-mediated dose limiting normal tissue toxicity. CDDP + WBH-mediated acute renal injury and CBDCA + WBH-mediated acute myelosuppression, as determined by blood urea nitrogen and peripheral blood cell counts, respectively, were not increased with the addition of TNF to either dual modality therapy. Histopathologically, addition of TNF produced no significant alterations in the kidney and the bone marrow as compared to CDDP + WBH or CBDCA + WBH. These data show that TNF enhanced the platinum + WBH-mediated antitumor effect without increasing normal tissue toxicity, suggesting that TNF may increase the therapeutic efficacy of CDDP or CBDCA combined with WBH.

Animals↗

PubMed1992

A role for calcium in regulating apoptosis in rat thymocytes irradiated in vitro.

Thymus-derived lymphocytes undergo death after gamma-irradiation via a pathway termed apoptosis, or programmed cell death. An early step in this pathway is the production of nucleosome-sized fragments of DNA. DNA fragmentation was used as the endpoint in these investigations to examine apoptosis in lymphocytes extracted from the rat thymus and irradiated in vitro. In unirradiated thymocytes the level of DNA fragmentation rose to 15% by the first hour of culture, where it remained approximately constant until the fifth hour. In contrast, thymocytes irradiated with a dose of 2.5 Gy exhibited a large and dramatic increase in DNA fragmentation beginning 2 h postirradiation. DNA fragmentation measured 6 h after irradiation was detected after as little as 0.25 Gy and reached a maximum of 90% with 10 Gy. Metabolic control of DNA fragmentation after irradiation was evidenced by the suppression of DNA fragmentation when thymocytes were incubated with cyclohexamide or actinomycin D. When gamma-irradiated thymocytes were incubated with the Ca2+ chelator EGTA, DNA fragmentation was reduced significantly. BAPTA-AM, a highly specific intracellular Ca2+ chelator, essentially eliminated DNA fragmentation in cells irradiated with 2.5 Gy and, unlike EGTA, eliminated the background level of fragmentation in unirradiated samples. Therefore, our data are consistent with the possibility that Ca2+ serves as a second messenger to induce DNA fragmentation in irradiated thymocytes, suggesting a common pathway for cells prompted to enter apoptosis from seemingly dissimilar interval events.

Animals↗

PubMed1992

Comparative in vitro studies of the potentiation of tumor necrosis factor (TNF)-alpha, TNF-beta, and TNF-SAM2 cytotoxicity by hyperthermia.

Hyperthermia can strikingly enhance tumor necrosis factor-alpha (TNF-alpha) cytotoxicity in vitro and in vivo. Other forms of TNF may have tumor therapeutic applications and their interaction with hyperthermia should also be assessed. We have compared the effect of heat on the in vitro cytotoxic response of murine L929 and EMT-6 and human T24 tumor cells to three TNF forms; recombinant human TNF-alpha, TNF-beta (lymphotoxin), and TNF-SAM2. A neutral red assay was used to measure toxicity at 18-20 h after initiating the heat treatment. TNF treatment preceded heating by 0-4 h or followed it by 2 h. Heating was done at 39 or 40.5 degrees C for 24 h, 40.5 or 42 degrees C for 1 h, or 43 degrees C for 1-1.5 h. We found that both TNF-beta and TNF-SAM2 toxicities, like that of TNF-alpha, were markedly enhanced by hyperthermia. Neither EMT-6 nor T24 cells responded consistently to any of these TNFs at heat doses up to 1 h at 43 degrees C, but an increment of only 15 min more at 43 degrees C sensitized EMT-6 cells and 1.5 h at 43 degrees C resulted in extensive EMT-6 cell killing. The T24 cells remained resistant except for variable responses at the highest TNF and heat doses. If TNF treatment was begun immediately before or 2 h after beginning to heat the EMT-6 cells, sensitization was reduced or eliminated, respectively, for all three TNF forms relative to protocols in which TNF was added 1, 2, or 4 h before heating.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Effects of calcium buffering on the synthesis of the 26-kDa heat-shock protein family.

We have reported on the effect of heat in C127 cells having various basal levels of the Ca(2+)-binding proteins calmodulin (CaM) or parvalbumin [Evans, Simonette, Rasmussen, Means, and Tomasovic, J. Cell. Physiol. 142, 615-627 (1990)]. These studies suggested that induction of the synthesis of 26-kDa heat-shock protein (hsp-26) depended on increased intracellular free Ca2+ [Ca2+]i and that induction was abrogated by increased Ca(2+)-binding capacity. To evaluate further the role of [Ca2+]i in mediating the response to hyperthermia and the potential for Ca(2+)-buffering to affect these processes, we loaded C127 parental cells with the Ca2+ chelators BAPTA or quin-2 (5 microM for 60 min) and then immediately heated the cells (30 min at 43 degrees C) and labeled them (3 h at 37 degrees C) with [3H]leucine. Measurements of [Ca2+]i with quin-2 and fura-2 showed that an increase in [Ca2+]i occurred with this heat dose, but that the quin-2 buffered that increase. Two-dimensional gels showed that cells loaded with BAPTA and quin-2 had a reduced rate of synthesis of the most basic (nonphosphorylated) hsp-26a isoform. The apparent synthesis of the more acidic isoforms (hsp-26b, hsp-26c) was less affected, but labeling studies with 32P showed this reflected continued accumulation of these phosphorylated isoforms, especially the most highly phosphorylated hsp-26c. Although it reduced hsp-26a synthesis, the temporary buffering of [Ca2+]i did not alter the subsequent expression of heat killing or the extent of thermotolerance significantly, possibly because phosphorylated hsp-26 was still generated. These data support the hypothesis that perturbations of [Ca2+]i directly modulate induction of hsp-26a synthesis.

Acclimatization↗

PubMed1990

Altered synthesis of the 26-kDa heat stress protein family and thermotolerance in cell lines with elevated levels of calcium-binding proteins.

Using a bovine papilloma virus-based vector, mouse mammary adenocarcinoma cells have been transformed to express elevated amounts of functional calmodulin (CaM) (Rasmussen and Means, 1987) and another Ca2(+)-binding protein, parvalbumin (PV) (Rasmussen and Means, 1989) that is not normally synthesized in these cells. Parental cells (C127) and cells transformed by the vector alone (BPV-1), the vector containing a CaM gene (CM-1), or the vector containing parvalbumin (PV-1) were used to study the effect of increased synthesis of Ca2(+)-binding proteins on heat-stress protein (HSP) synthesis and cell survival following heating at 43 degrees C. The induction, stability, and repression of the synthesis of most HSPs after 43 degrees C heating was not significantly affected by increased amounts of Ca2(+)-binding proteins, but the rate of synthesis of all three isoforms of the 26-kDa HSP (HSP26) was greatly reduced. C127 cells, which have about one half as much CaM as do BPV-1 cells, synthesized the most HSP26. CM-1 cells, which have more than fourfold higher levels of CaM than do BPV-1 cells, had a rate of synthesis of HSP26 approaching that of unheated cells. BPV-1 cells, with a two-fold increase in CaM, were intermediate in HSP26 synthesis. This effect on HSP26 synthesis may be largely related to the Ca2(+)-binding capacity of CaM rather than to a specific CaM-regulated function, since PV-1 cells also showed reduced rates of HSP26 synthesis. Survival experiments showed that reduced HSP26 synthesis in cells with increased amounts of Ca2(+)-binding proteins did not significantly alter intrinsic resistance to continuous 43 degrees C heating. Thermotolerance was not reduced and appeared to develop more rapidly in CM-1 and PV-1 cells. These results suggest that (1) the signal for HSP26 synthesis can be largely abrogated by elevated Ca2+ binding protein levels, and (2) if these HSPs are involved in thermotolerance development, that function may be associated with intracellular Ca2+ homeostasis.

Animals↗

PubMed1990

Affinity isolation of heat-shock and other calmodulin-binding proteins following hyperthermia.

The interaction of calmodulin (CaM) with heat-shock and other binding proteins was studied in rat adenocarcinoma cells. Cells were equilibrium-labeled for 48 h prior to heating for 1 h at 43 degrees C, or pulse-labeled for 2 h at 37 degrees C after heating, to monitor the effect of heat on the affinity of CaM-binding proteins synthesized under these conditions. A CaM antagonist shown to sensitize to heat killing, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], was used in competition assays to help monitor any changes in affinity. We found that heating tended to reduce the CaM-binding of proteins synthesized before heating relative to their 37 degrees C controls and proteins synthesized after heating tended to have increased binding relative to their respective controls. Members of the heat-shock protein (hsp) 90-, 70-, and 26-kDa families were among the proteins that bound to CaM and were eluted by W-7. The peak elution fractions for the hsp's and other cellular proteins varied, but hsp-70 eluted in the early fractions. The hsp-70 family was also found to be among a number of W-7-binding proteins. We conclude that the assumption that CaM antagonists potentiate killing of heated cells solely by competing nonspecifically for CaM-binding protein sites on CaM does not explain the process completely. These antagonists could also act by competing for CaM-binding sites with specific proteins whose interaction with CaM is important for survival following heating, or by directly binding to other proteins whose function is important for survival and inhibiting their activity. We do not have sufficient data to discern the predominant mechanism among these possibilities, but we believe all are likely to occur in heated cells and speculate that inhibition of the functions of the hsp-70 family is important in several of these antagonist actions.

Adenocarcinoma↗

PubMed1989

Hyperthermic modulation of respiratory inhibition factor- and iron releasing factor-dependent macrophage murine tumor cytotoxicity.

Hyperthermia modulated the cytotoxic activities of murine macrophages cocultured with EMT-6 tumor cells, altered the production of the monokines RIF (respiratory inhibition factor) and FeRF (iron-releasing factor) by these effector cells, and perturbed the activities of these monokines against EMT-6 cells. Cytotoxic activities of heated murine macrophages activated by Bacillus Calmette-Guérin were inhibited by heat doses of 40.5 degrees C for greater than or equal to 1 h if heating preceded triggering by the endotoxin lipopolysaccharide; however, cytotoxic activities were better retained if triggering preceded heating by 2 h. Treatment-sequence dependencies were also found in the secretion of some monokines that participate in these macrophage cytotoxic effects. Secretion of both RIF and FeRF by macrophages was slightly augmented or at least better retained if triggering of macrophages occurred several hours before heating for 1 h at 40.5-43 degrees C or for 24 h at 39-40.5 degrees C. If heating was nearly simultaneous with triggering or preceded it by several hours, there was a dose-dependent decrease in monokine secretion. The sensitivity of tumor cell targets to these monokines was also modified by the treatment sequence. When EMT-6 cells were treated with RIF- or FeRF-containing macrophage conditioned supernatants 1 h before heating at 40.5-43 degrees C, the RIF-treated cells became sensitized and the FeRF-treated cells retained better cytotoxic response than if cells had been treated 1 h after heating. Chronic heating for 24 h at 39-40.5 degrees C showed less dependence of tumor cell response on treatment sequence. Similar observations have recently been made in our laboratory for the interaction of tumor necrosis factor cytotoxic pathways with hyperthermia (Klostergaard et al., J. Biol. Response Modif., in press, 1989; Tomasovic et al., Int. J. Hyperthermia, in press, 1989). Those results and the present observations with the monokines RIF and FeRF support our hypothesis that the cytotoxic actions of macrophages and the cytotoxicity of endogenously added monokines can be augmented by appropriately constructed sequences combining hyperthermia with either macrophage priming/triggering or monokine treatment of tumor cells.

Adenocarcinoma↗

PubMed1989

Hyperthermic modulation of macrophage-tumor cell interactions.

Hyperthermia in the febrile (less than or equal to 41 degrees C) or tumor therapeutic (greater than or equal to 42 degrees C) ranges is known to alter tumor-host interactions: there are reports of either inhibitory or enhancing effects on tumor metastasis and various host defense mechanisms. Historically, this has been an area of conflicting and often anecdotal reports, and there are still significant gaps in our knowledge of the effects of temperature on tumor-host interactions. However, we believe that the tools are now available to further our understanding of the complex relationships between febrile episodes or therapeutically applied heat and various tumor-host cytotoxic mechanisms, and that potentially important and exploitable relationships can be defined. In this review we give an overview of the current status of this field and the factors that have shaped it. We also describe our recent experimental work with macrophages and their monokines, primarily tumor necrosis factor (TNF), which we feel offers new scientific and clinical opportunities for future studies.

Animals↗

PubMed1989

Application of MR spectroscopy to the study of tumor biology.

Though MR spectroscopy has long been used to analyze the structure of organic compounds in solution, interest in applying it to the study of biologic systems has been slow in evolving because of past problems with spectral resolution in solids and gels. Renewed interest in this area has been stimulated both by the rapid growth of MR imaging as a clinical tool as well as by improvements in MR spectrometer design and use of sophisticated pulse sequences which have greatly improved the analysis of molecular composition. This review specifically focuses on the application of MR spectroscopy to studying the biology of malignant cells. The bulk of MR studies in this area to date have involved either 1H or 31P spectroscopy. Several investigators have now demonstrated that 1H spectra can be used to distinguish both animal and human tumors of differing metastatic properties. Preliminary data suggest that these spectral differences result in part from differences in cell surface glycoproteins and/or glycolipids between cells of low and high metastatic potential. Many of these molecules can absorb cell water potentially affecting T1 of cell water by their relative concentrations. Prolonged T2 relaxation times have been associated with some spectral peaks which distinguish cells of differing metastatic potential. The findings may partly explain why tumors have the prolonged T1 and T2 relaxation times seen in proton MR imaging. Other 1H MR spectroscopic studies suggest that there are detectable differences in plasma lipids in patients with a variety of malignancies compared to normal controls, suggesting possible utility as a screening test.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Hyperthermic modulation of tumor necrosis factor-dependent monocyte/macrophage tumor cytotoxicity in vitro.

Tumor necrosis factor (TNF) production by human peripheral blood monocytes and murine bacillus Calmette-Guerin-activated peritoneal macrophages was strongly influenced by acute hyperthermia. If hyperthermia was administered simultaneously with or preceding lipopolysaccharide triggering, production was severely ablated by 42 degrees and 43 degrees C treatments; however, if triggering preceded heating by at least 90 min, production was either unaffected or markedly enhanced. A somewhat similar pattern was reflected with chronic heating. TNF production by murine macrophages was inhibited with 39 degrees C heating and completely blocked by 40.5 degrees C treatment, if triggering coincided with the initiation of hyperthermia. However, augmentation of production occurred with either of these temperatures if triggering preceded hyperthermia by as little as 90 min. Human monocytes demonstrated greater resistance to the deleterious effects of coincident triggering and heating with respect to TNF secretion than the rodent effectors, but the response was otherwise very similar. The TNF-sensitive phenotype of the L929 cell could be augmented by chronic or acute hyperthermia, markedly so with a 43 degrees C treatment. The TNF-resistant phenotype of the EMT-6 cell could be reversed by chronic heating at 40.5 degrees C, or by acute heating at 43 degrees C, but only if the latter followed TNF treatment. These results reflect important regulatory controls of TNF production and responses in tumor cells which are susceptible to hyperthermia manipulation.

Animals↗

PubMed1989

Neutral red uptake and clonogenic survival assays of the hyperthermic sensitization of tumor cells to tumor necrosis factor.

Vital dye uptake and postfixation dye assays have recently been used to examine the interaction between short-term (24-48 h) exposures to the monokine, tumor necrosis factor (TNF), and hyperthermic treatments with the finding that synergistic increases in cytotoxicity occurred. However, survival measured by these short-term dye assays is not necessarily closely related to eventual loss of clonogenic capacity. Treatment-induced growth delays, delayed cytotoxic effects, or perturbations of vital dye sequestration mechanisms could result in a different measurement of surviving fraction than given by a clonogenic assay. In this study we directly compared the neutral red vital dye uptake and clonogenic survival assays and confirmed in both assays that TNF-sensitive (L-929) and TNF-resistant (EMT-6) phenotypes show greatly reduced survival when treated with combined recombinant human TNF (1.0-0.0005 micrograms/ml) and hyperthermia (1-2 h at 43 degrees C). Moreover, we confirmed that sensitization of the TNF-resistant EMT-6 cells was largely dependent on monokine treatment before hyperthermia and was reduced by the reverse sequence. The greatest sensitization of TNF-responsive L-929 cells also occurred when TNF treatment preceded heating. These results for clonogenic survival are consistent with the hypothesis that hyperthermia used in combination with TNF in vivo is more cytotoxic than TNF or hyperthermia separately.

Animals↗

PubMed1987

Proton NMR examination of tumor cells of high or low metastatic potential.

Three rat 13762NF mammary adenocarcinoma clones and cell lines of different metastatic potentials (MTLn3, MTC, and MTPa) were studied for their proton nuclear magnetic resonance spectral characteristics as intact cells in vitro and after chloroform/methanol, neuraminidase, or ethanol treatments. The intact-cell spectral characteristics of the highly metastatic tumor cell clone MTLn3 were clearly distinguished from the less metastatic clone MTC or the parental MTPa cell line on the basis of spectral peaks in the range of 0.9 to 1.45 p.p.m. broad peaks near 2.0 p.p.m., and peaks in the range of 2.75 to 3.2 p.p.m. Glycoproteins are among the molecules known to have resonances in these upfield spectral regions, and these tumor cell subpopulations have previously been shown to possess characteristic quantitative differences in cell surface, metastasis-associated glycoproteins. Treatment of the cells with neuraminidase or ethanol, or extraction with chloroform/methanol increased spectral detail and also revealed characteristic differences in spectral peaks between the tumor cell subpopulations. The identity of the cellular components responsible for these spectral characteristics are unknown, but some clearly arise from differences in the extractable lipids present in the tumor cell subpopulations. Further study will be required to determine if the spectral differences described in this preliminary report are directly related to the known biochemical characteristics of the highly metastatic clone, and if the observations have general relevance to metastatic potential or are a singular feature of these cells. However, these initial results suggest that manipulation of factors which allow unmasking of spectral detail combined with the use of prescribed tumor cell subpopulations may aid in using proton NMR to identify and define biochemical or structural differences related to the metastatic potential of tumor cells.

Adenocarcinoma↗