Differences in oxygen-dependent regulation of enzymes between tumor and normal cell systems in culture

Metabolic studies in tumor cells have indicated that bioenergetic regulatory mechanisms geared to acute changes in oxygen availability are abnormal. In the present studies we have examined bioenergetic adaptations to chronic oxygen depletion in culture maintained tumor cells in comparison to normal cell lines. Activities of two key glycolytic enzymes (pyruvate kinase (PyKI) and phosphofructokinase (PFK)) were measured in two tumor cell lines (fibrosarcoma (FS) and Hela) and two normal cell lines (rat lung fibroblasts (RLF) and WI-38) maintained in culture for up to 96 hours under aerobic (PO2 approximately 140) and hypoxic PO2 approximately 15) conditions. Exposure to low O2 tensions for 96 hours resulted in significant increases in PyKi and PFK in both RLF and WI-38, ut did not alter activities of these enzymes in either FS or HeLa cell systems. Activities of two enzymes involved in O2 metabolism (cytochrome oxidase (CyOx) and superoxide dismutase (SOD) were also measured in the two tumor cell lines and in RLF. chronic hypoxia significantly decreased the activities of CyOx and SOD in RLF cell systems but did not alter the activities of these enzymes in the tumor cells. In these studies, the tumor-derived cell lines do not demonstrate specific enzymatic responses to sustained oxygen depletion in vitro noted in normal cell systems, suggesting significant abnormalities in regulatory mechanisms geared to chronic changes in molecular O2.     

THE DISCRETE–TIME KINETIC MODEL ANALYSIS OF DNA CONTENT DISTRIBUTIONS IN EXPERIMENTAL TUMOUR CELLS

A method was developed to analyse and characterize FMF measurements of DNA content distribution, utilizing the discrete time kinetic (DTK) model for cell kinetics analysis. The DTK model determines the time sequence of the cell age distribution during the proliferation of a tumor cell population and simulates the distribution pattern of the DNA content of cells in each age compartment of the cell cycle. The cells in one age compartment are distributed and spread into several compartments of the DNA content distribution to allow for different rates of DNA synthesis and instrument dispersion effects. It is assumed that the DNA content of cells in each age compartment has a Gaussian distribution. Thus, for a given cell age distribution the DNA content distribution depends on two parameters of the cells in each age compartment: the average DNA content and its coefficient of variation. As the DTK model generates the best fit DNA content distribution to the FMF measurement data, it enables one to estimate specific values of these two parameters in each stage of the cell cycle and to determine the fraction of cells in each cycle phase. The method was utilized to fit FMF measurements of DNA content distributions and to analyse their relationship to the cell kinetic parameters, namely cell loss rate, cell cycle times and growth fraction of exponentially growing Chinese hamster ovary cells in vitro and, also, with a wide range of coefficients of variation, of the L1210 ascites tumour during the growth period.     

Glycosphingolipid deficiency affects functional microdomain formation in Lewis lung carcinoma cells

In view of the increasing evidence that gangliosides in membrane microdomains or rafts are closely associated with various signal transducing molecules including Src family kinases, we compared rafts in two subclones of 3LL mouse lung carcinoma cell line, J18 and J5, characterized by high and very low GM3 ganglioside contents, respectively. Rafts were isolated from cell lysates as low density detergent-insoluble microdomains (DIM) by sucrose density gradient centrifugation. J5 and J18 cells expressed comparable amounts of Src family kinases and the majority of Src kinases in both clones were concentrated in their DIMs, suggesting that GM3 is not necessary for DIM localization of Src kinases and there is no direct interaction between Src and GM3. However, the Src kinases were eliminated from DIMs after depletion of the major neutral GSLs of J5 cells, glucosylceramide and lactosylceramide, by an inhibitor of glucosylceramide synthase (D-PDMP), indicating that GSLs in general are required for Src kinase association to DIM. J5 and the D-PDMP-treated J5 cells had very similar DIM protein profiles and moreover cholesterol and sphingomyelin in the GSL-depleted cells were enriched in DIM similar to the untreated control cells. Interestingly, the levels of tyrosine-phosphorylated DIM proteins and cell proliferation of J5 cells were much lower than those of J18 cells, suggesting that GM3 might be involved in tyrosine phosphorylation of DIM proteins required for cell growth. Thus, our data suggest that GSLs are essential for functional raft formation.     

The influence of different flow velocities on tumor cell survival in micropore filters.

Earlier studies have shown a lower degree of lodgement and early survival of tumor cells in muscle than in liver after infusion via the femoral artery and portal vein, respectively. A possible explanation to this difference might be that the tumor cells are mechanically destroyed, and thus die more rapidly in muscle because they enter this capillary network at a much higher flow velocity. In the present study, the effect on early tumor cell (rat fibrosarcoma) survival of a high and a low flow velocity/deformation rate was evaluated in micropore (5 microns) filters, using isotope (Cr51) technique. These experiments were combined with scanning electron microscopic (SEM) analyses. The filter experiments showed no significant differences in the rate of cell death in the filters between tumor cells subjected to high or low deformation rates, and there were no qualitative differences in tumor cell appearance in the SEM study. It is, therefore, concluded that the difference in tumor cell lodgement and survival between muscle and liver is not primarily caused by differences in the rate of cell deformation upon entry of the organ capillary network.     

Protein synthesis, ribosomal protein S6 phosphorylation in vitro and the effects of amiloride: SDS gel electrophoresis studies in the Yoshida ascites tumor (AH 130) grown in vivo

Cell-free cytosolic extracts from the Yoshida (AH 130) rat ascites hepatoma cell line, grown in vivo, showed high ribosomal protein S6 kinase activity in vitro, as measured by transfer of 32P to exogenous 40S rat liver ribosomal subunits, in both exponential growing and stationary phase cells. A significant decrease of protein synthesis (3H-leucine incorporation into total cell protein) was found to occur in cells reaching the stationary phase of growth, suggesting that S6 phosphorylation was not tightly coupled to the rate of the intraperitoneal cell growth and of protein synthesis in these tumor cells. When the cell-free cytosolic extracts were prepared from cells exposed to amiloride, at concentrations that inhibit the Na+/H+ exchange, a decrease of S6 kinase activity was observed only in exponential growing cells, suggesting the possibility of coupling of the Na+/H+ exchange with phosphorylation of intracellular proteins in these tumor cells. Actually, stationary phase cells showed unchanged S6 kinase activity under the same conditions, possibly due to the extremely low Na+/H+ exchange activity, previously demonstrated (Cell Biol. Int. Rep., 1985, 9, 1017-1025). The present experiments support the hypothesis that the regulation of protein synthesis is not tightly coupled to phosphorylation-dephosphorylation cycles, at least of ribosomal protein S6, in cells characterized by a rather uncontrolled growth such as the Yoshida (AH 130) rat ascites hepatoma. In this connection, an elevated degree of protein phosphorylation, such as that of the ribosomal protein S6, could be a general phenomenon of neoplastic transformation.     

Liquid ordered phase in cell membranes evidenced by a hydration-sensitive probe: Effects of cholesterol depletion and apoptosis

Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells.     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

Sialylation of 3-methylcholanthrene-induced fibrosarcoma determines antitumor immune responses during immunoediting

Sialylation of tumor cells is involved in various aspects of their malignancy (proliferation, motility, invasion, and metastasis); however, its effect on the process of immunoediting that affects tumor cell immunogenicity has not been studied. We have shown that in mice with impaired immunoediting, such as in IL-1α(-/-) and IFNγ(-/-) mice, 3-methylcholanthrene-induced fibrosarcoma cells are immunogenic and concomitantly bear low levels of surface sialylation, whereas tumor cells derived from wild type mice are nonimmunogenic and bear higher levels of surface sialylation. To study immune mechanisms whose interaction with tumor cells involves surface sialic acid residues, we used highly sialylated 3-methylcholanthrene-induced nonimmunogenic fibrosarcoma cell lines from wild type mice, which were treated with sialidase to mimic immunogenic tumor cell variants. In vivo and in vitro experiments revealed that desialylation of tumor cells reduced their growth and induced cytotoxicity by NK cells. Moreover, sialidase-treated tumor cells better activated NK cells for IFN-γ secretion. The NKG2D-activating receptor on NK cells was shown to be involved in interactions with desialylated ligands on tumor cells, the nature of which is still not known. Thus, the degree of sialylation on tumor cells, which is selected during the process of immunoediting, has possibly evolved as an important mechanism of tumor cells with low intrinsic immunogenicity or select for tumor cells that can evade the immune system or subvert its function. When immunoediting is impaired, such as in IFN-γ(-/-) and IL-1α(-/-) mice, the overt tumor consists of desialylayed tumor cells that interact better with immunosurveillance cells.     

The influence of different flow velocities on tumor cell survival in micropore filters

Earlier studies have shown a lower degree of lodgement and early survival of tumor cells in muscle than in liver after infusion via the femoral artery and portal vein, respectively. A possible explanation to this difference might be that the tumor cells are mechanically destroyed, and thus die more rapidly in muscle because they enter this capillary network at a much higher flow velocity. In the present study, the effect on early tumor cell (rat fibrosarcoma) survival of a high and a low flow velocity/deformation rate was evaluated in micropore (5 μm) filters, using isotope (Cr⁵¹) technique. These experiments were combined with scanning electron microscopic (SEM) analyses. The filter experiments showed no significant differences in the rate of cell death in the filters between tumor cells subjected to high or low deformation rates, and there were no qualitative differences in tumor cell appearance in the SEM study. It is, therefore, concluded that the difference in tumor cell lodgement and survival between muscle and liver is not primarily caused by differences in the rate of cell deformation upon entry of the organ capillary network.     

Relationship between energy status, hypoxic cell fraction, and hyperthermic sensitivity in a murine fibrosarcoma

The energy status, radiobiological hypoxic cell fraction, and hyperthermic sensitivity of a spontaneous murine fibrosarcoma, FSa-II, have been evaluated as a function of tumor size. Tumors were evaluated over the size range of 70 to 800 mm3. The concentration of the high-energy phosphate reservoir creatine phosphate progressively decreased by a factor of 5 with increasing tumor volume, and was matched by an increase in creatine. The concentration of ATP also decreased with increasing tumor size, although this decrease was substantially less pronounced. The sum of ATP, ADP, and AMP did not vary with tumor size, suggesting that the necrotic fraction remained constant. The decrease in energy status occurred in parallel with an increase in the size of the hypoxic cell fraction and with increasing thermal sensitivity. The results suggest that energy status may be an important modifier of hyperthermic sensitivity in vivo and reflect tissue oxygen concentration.     

A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Tumor initiating cells: Development and critical characterization of a model derived from the A431 carcinoma cell line forming spheres in suspension

To investigate the tumor fraction with cancer stem/tumor initiating cell (CSC/TIC) characteristics, we tested the human cervical carcinoma cell lines A431, Caski and SiHa, by growth as non-adherent spheres in specific media and aldehyde dehydrogenase (ALDH) enzymatic activity. A good correlation between the two parameters was observed and the highest levels were observed in A431 cell line that was selected for characterization of the CSC/TIC fraction. A431 parental cells already displayed characteristics common to CSC/TIC, such as sphere forming efficiency, adherent holoclone formation and high ALDH activity. Non-adherent spheres maintained or increased these properties, and, in particular, ALDH-positive fraction increased from 46 to 65% and a transient induction of stem cell markers such as Nanog, Nestin and Oct4 was observed. Furthermore, a significant increase of paraclone forming cells was observed, suggesting that differentiation took place inside sphere cell populations. As compared to parental cells, spheres were characterized by: (1) a ten-fold higher verapamil-sensitive side population fraction; (2) the appearance of a podoplanin-positive subpopulation characterized by a small cell size; (3) the ability to propagate tumors in nude mice at a lower cell dose. The global gene expression analysis demonstrated a strong and reversible modulation of 'sphere' phenotype in comparison to parental and sphere cells re-induced to adherent conditions. All together our results indicated that the growth of A431 cells as a non-adherent sphere was not sufficient by itself to define a stem-like population, but it was essential for the emergence of a small population of tumor cells with CSC properties.     

The effect of hyperglycemia on the tumor response to irradiation given alone or in combination with hyperthermia

The effect of hyperglycemia (elevated blood glucose level) on the response of a murine tumor to irradiation given alone or in combination with hyperthermia was studied. Tumors were early generation isotransplants of a spontaneous C3H/Sed mouse fibrosarcoma, FSa-II. Single-cell suspensions were transplanted into the foot, and irradiation was given when each tumor reached an average diameter of 7 mm. Following irradiation, the tumor growth time to reach 1000 mm3 was studied and the dose-response curve between the tumor growth time and radiation dose was fitted. Preadministration of glucose increased the size of the hypoxic and chronically hypoxic cell fractions without altering the slope of the dose-response curve where the chronically hypoxic cell fraction is determined as the fraction of cells which were not oxygenated under hyperbaric oxygen conditions. Hyperthermia given prior to irradiation enhanced the tumor response to irradiation, but simultaneously increased the size of the hypoxic and chronically hypoxic cell fractions. Similar results were observed following hyperthermia given after irradiation. When hyperthermia at 43.5 degrees C was given 24 h before irradiation, the size of the hypoxic cell fraction increased with increasing treatment time, while a substantial decrease in the chronically hypoxic cell fraction was observed. Administration of glucose 60 min before hyperthermia further increased the size of the hypoxic cell fraction. Possible mechanisms explaining why glucose administration increases the hypoxic cell fractions are discussed.     

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of particles in the smaller peak (6-11 microm diameter) closely correlated with the fractions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells.     

Prophylactic anti-tumor immunity against a murine fibrosarcoma triggered by the Salmonella type III secretion system

The potential of an attenuated Salmonella enterica serovar Typhimurium strain as a prophylactic anti-tumor vaccine against the murine fibrosarcoma WEHI 164 was evaluated. Tumor cells were transfected with the DNA sequence encoding the MHC class I-restricted peptide p60(217-225) from Listeria monocytogenes. BALB/c mice received a single orogastric immunization with Salmonella that translocates a chimeric p60 protein via its type III secretion system. Mice were subsequently challenged subcutaneously with p60(217-225)-expressing WEHI cells. In vivo protection studies revealed that 80% of these mice remained free of the fibrosarcoma after challenge, whereas all animals of the non-vaccinated control group did develop tumor growth. In further experiments, the distribution of tetramer-positive p60(217-225)-specific effector and memory CD8 T cells after Salmonella-based immunization and tumor application was analyzed. Costaining with CD62L and CD127 revealed a predominance of p60-specific central memory and effector memory CD8 T cells in spleens, whereas in blood samples the majority of p60-specific lymphocytes belonged to effector and effector memory CD8 T cell subsets. This is the first report demonstrating that a bacterial type III secretion system can be used for heterologous antigen delivery to induce cytotoxic effector and memory CD8 T cell responses resulting in an efficient prevention of tumor growth.     

Cell surface changes correlated with density-dependent growth inhibition. Glycosaminoglycan metabolism in 3T3, SV3T3, and con A selected revertant cells.

A 35SO4-labeling/chromatography technique has been developed which facilitates quantitation of sulfated glycosaminoglycan (GAG) synthesis in mammalian cell cultures. The technique has been used to compare sulfated GAG biosynthesis, degradation, and turnover in three related cell lines with differing degrees of density-dependent inhibition of growth in vitro (Balb/c 3T3, SV3T3, and SV3T3 revertant cells). Viral transformation of Balb 3T3 cells is accompanied by a 2-5-fold decrease in cell associated sulfated GAG. SV3T3 revertant cells, which show partial reversion to low saturation density in vitro, show a 2.5-8-fold increase in cell-associated sulfated GAG compared to the parental SV3T3 cells from which they were selected. In addition, the distribution of 35SO4 and [3H]glucosamine among the different GAG species produced by SV3T3 revertant cells reverts so that it is similar to the distribution characteristic of untransformed 3T3 cells rather than SV3T3 cells. Mild trypsin treatment of 35SO4-labeled cells removed 68-84% of the cellular sulfated GAG, suggesting that at least this proportion of the total cellular sulfated GAG was located at the cell periphery. Removal of 35SO4-labeled cells from the Petri dish with a Ca2+ selective chelating agent revealed a fraction of the sulfated GAG that remained tightly bound to the Petri dish. A higher proportion of the total cell-associated sulfated GAG remained attached to the Petri dish in cultures of untransformed and revertant cells compared to that present in cultures of transformed cells. A role for sulfated GAG in density-dependent growth inhibition of fibroblast cultures is proposed and discussed in the light of the data obtained.     

Soluble tumor necrosis factor receptor type I enhances tumor development and persistence in vivo

Secretion of human soluble tumor necrosis factor receptor type I (sTNFRI) by the mouse fibrosarcoma cell line, L929, previously has been demonstrated to confer resistance to in vitro lysis by TNF and to LAK- and CTL-mediated cytolysis. These findings suggest that, in vivo, sTNFRI contributes to tumor survival by inhibiting these immunologic mechanisms. To evaluate this hypothesis, we compared the growth of sTNFRI-secreting L929 cells with that of the unmodified parental fibrosarcoma in an in vivo mouse transplantation model. Secretion of sTNFRI by L929 cells markedly enhanced their tumorigenicity and persistence in syngeneic recipients. This benefit was abrogated by sTNFRI-neutralizing antibodies induced by immunization prior to tumor challenge. These data demonstrate that sTNFRI directly influences tumor formation and persistence in vivo and suggest the selective removal and/or inactivation of sTNFRI as a promising new avenue for cancer immunotherapy.     

Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in prebeta-HDL generation

The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce pre(beta)-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process.     

Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in preβ-HDL generation

The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce preβ-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process.     

Anti-tumor activity of recombinant tumor necrosis factor on mouse fibrosarcoma in vivo and in vitro

The experiments presented were designed first to determine the effects of rTNF on the methylcholanthrene-induced fibrosarcoma (FSA-1) in C3H/JSed mice and second to determine whether the observed effects are the result of direct action by rTNF on the tumor or whether rTNF acts as a mediator of other effector mechanisms. Mice received syngeneic FSA-1 fibrosarcoma cells either s.c. or i.v. in order to evaluate growth of transplantable solid tumor or lung metastases, respectively. The range of dosages, from 10(2) to 2 x 10(5) U of rTNF, was administered i.v. at different intervals after the tumor cell injection. Early injection of 10(3) to 10(4) U of rTNF reduced the growth of s.c. injected tumor and the number of lung metastases in i.v. injected mice. In both cases, survival of mice was also prolonged. However, in vitro treatment of FSA-1 tumor cells with rTNF did not result in the reduction of their proliferating activity after injection into mice, although direct cytostatic and moderate cytotoxic activity of rTNF in vitro was demonstrated. To identify whether other cellular mechanisms are involved in the effects observed in vivo, the anti-tumor activity of rTNF-treated spleen cells was evaluated in vitro using a 75Se release assay. Whereas nontreated spleen cells demonstrated very low cytotoxic activity in this system, the cells from rTNF-treated mice showed marked increase in the cytotoxicity against syngeneic tumor cells. These results suggest that the anti-tumor activity of rTNF represents a combination of its direct effect on tumor cells and indirect effects involving host immune mechanisms.