Effect of hyperthermia on AKR lymphoma variants differing in degree of malignancy.

The effect of hyperthermic treatment on AKR lymphoma cells of varying malignancy was investigated. Tumor cells were pretreated at 37 or 43 degrees C and then injected to mice. The effect on the highly malignant variant, TAU-38, was compared to that on the low-malignancy variant, TAU-39, following both subcutaneous (s.c.) and intravenous (i.v.) inoculation. Hyperthermia showed no effect on the TAU-39 variant following s.c. inoculation on the primary tumors or mice survival, but the TAU-38 variant exhibited a significant delay of tumor appearance following treatment, namely, decreased tumor size and increased life span. Following i.v. inoculation, in both variants, hyperthermia caused a significant decrease in metastatic spread and an increased life span. We conclude that hyperthermia, in addition to exerting a greater effect on the high-malignancy variant, acts at the late phases of metastasis. Hyperthermia might therefore have a place in the management of cancer in its advanced disseminated phase.

Apoptosis and cell proliferation capacity in AKR lymphoma malignancy variants.

Dysregulation in apoptotic cell death has recently emerged as a factor in tumorigenesis, but its effect in tumor progression is not yet established. In the present study we evaluated the levels of proliferative and apoptotic cell fractions in a T-cell lymphoma tumor progression model. We compared these features and the expression of apoptosis-related genes in primary tumors of several AKR lymphoma malignancy variants. According to DNA flow cytometry, a considerable proportion of cells (35-40%) was in the proliferative (S + G2/M) phase in all variants, but a slight augmentation with increasing malignancy was noted. Apoptotic cell content was, unexpectedly, the lowest in the less malignant variant. This might be due to the higher content in macrophages observed in this variant, which possibly partly eliminated apoptotic bodies. We found an increase in bcl-2 level with increasing malignancy that was probably counterbalanced by the simultaneous increase observed in the Fas receptor.

Macrophage-recognized molecules of apoptotic cells are expressed at higher levels in AKR lymphoma of aged as compared to young mice.

While a direct relation between aging and tumorigenesis is well established, a slower tumor progression rate was reported in old as compared to young cancer patients. The mechanisms responsible for the less aggressive behavior of tumors in the aged, are largely unknown. We have recently shown an increase in apoptotic cell death in tumors derived from aged as compared to young animals in the AKR lymphoma. This was shown by DNA flow cytometry and by the ladder type DNA fragmentation in agarose gel electrophoresis. Analysis of the expression of genes involved in apoptosis in tumors derived from young and old animals showed a lower bcl-2 expression in those from the aged. The Fas antigen, on the contrary, displayed higher expression levels on lymphoma cells derived from old than on those from young mice. Apoptotic cells are recognized and phagocytosed mainly by macrophages. One molecular property of apoptotic cells which is recognized by macrophages is a loss in cell surface sialic acid concomitantly uncovering galactose residues. While comparing the "eat me status" phenotype of the tumor cells derived from young and aged animals, by the use of lectins recognizing sialic acid and galactose residues, FACS analysis showed a decrease in cell surface sialic acid and a gain in galactose residues in aged as compared to young mice. Moreover, Western blot analysis showed that a 130 Kda sialylated membrane glycoprotein was expressed at a lower level in tumors from the old as compared to young mice. Our results, at both the cellular and molecular levels, particularly with regard to molecules recognized by macrophages, indicate that increased apoptotic cell death in tumors from old as compared to those from young animals constitutes, as we have previously suggested, one of the mechanisms of the age-related decrease in tumor progression rate.

Sensitivity to macrophages decreases with tumor progression in the AKR lymphoma.

Resistance to immune reactions, innate or acquired, may be one of the mechanisms responsible for the progression of tumors. We have, indeed shown higher numbers of macrophages surrounding low- as compared to high-malignancy cells. In the present study we examined the level of cell surface molecules known to determine sensitivity to macrophages, namely galactose (GAL) and sialic acid (SA) residues. A histochemical assay for identification of SA by electron microscopy showed a higher cell surface content on metastatic (MT) than on primary (PT) tumor cells. The FACS data seen with fluorescent lectins showed a higher binding of Sambucus nigra agglutinin, which identifies SA attached to terminal GAL in -2.6 or -2.3 linkage, in MT than in PT cells. Binding of Maakia amurensis lectin (MAL-1), which identifies SA at position 3 of GAL, showed that the MT cells contain two subpopulations, one binding more MAL-1 and another less. Cell sorting showed a more aggressive behavior of the first population. The comparison of Peanut agglutinin (PNA) binding, which identifies GAL, demonstrated a decreased amount of PNA receptors in MT as compared to PT cells. Western blot analysis of the membranal proteins with different lectins, identified 3 sialylated glycoproteins. The 88 kDa glycoprotein had no significance for metastatic potential. The 130 kDa glycoprotein was higher in MT than on PT cells. The 220 kDa glycoprotein was practically present only on MT cells. The tendency observed was of a higher level of membranal glycoconjugates terminally sialylated with subterminal galactose residues, inMT cells as compared to PT cells. This may explain the recently found decrease in apoptotic cell death with increasing aggressiveness of the AKR lymphoma and suggests a lower sensitivity to macrophages with tumor progression. Treatment based on the reduction in sialic acid content might render the tumor cells more vulnerable to macrophages. We found, indeed, that Wheat germ agglutinin (WGA) injected in vivo, exerted an inhibitory effect on growth of the lymphoma. We found moreover that WGA-treated tumor cells were more sensitive than nontreated cells to macrophages in vitro.

Release from apoptosis correlates with tumor progression in the AKR lymphoma.

Disturbance of apoptosis is an established factor in tumorigenesis. The role of apoptosis in tumor progression is not yet clear. In the present study we compared the tendency to spontaneous apoptosis (and the proliferative capacity) of tumor cells derived from primary (PT) and metastatic tumor (MT) cells of several AKR lymphoma variants. Apoptosis-related gene expression was also compared. Our results indicate that release from apoptosis has a role in the tumor progression of this T cell lymphoma. At the cellular level, a markedly lower apoptotic tendency was observed in MT than in PT cells. The existence of macrophages only in PT also supports the presence of apoptotic cells in local but not in MTs. By contrast, proliferative capacity does not determine tumor aggressiveness in this system. At the molecular level, we found a higher staining intensity for bcl-2 in MT than in PT cells, suggesting that bcl-2 might be responsible for the reduced apoptosis in MT compared to PT cells. Evidence for p53 overexpression was found in the MT cells of one of the variants but in none of the PT. Comparison of Fas receptor, unexpectedly showed an increased expression in MT versus PT cells, possibly indicating resistance to Fas-induced apoptosis in the MT cells.

Apoptotic cell death and related gene expression in metastatic tumors of AKR lymphomas of varying malignancy.

Resistance to apoptosis may be related to tumor progression, due to the implications it might have on both tumor mass and genetic instability. We compared the tendency to spontaneous apoptosis and the proliferative capacity of metastatic growths of several AKR lymphoma variants (TAU-45, TAU-47, TAU-44, TAU-33, TAU-42 and TAU-46, in the order of increasing metastatic potential). We further compared the expression of several apoptosis-related genes. Cell proliferative capacity did not appear to determine malignant behavior since, on the whole, a decrease in S + G2M fraction was observed with increasing malignancy. Sensitivity to apoptotic cell death decreased with increasing malignancy when comparing the TAU-45, TAU-47, TAU-44 and TAU-33 variants, suggesting a role of reduced apoptosis in this T-cell lymphoma. An increase in Bcl-2 content with increasing aggressiveness among these variants, implicates this protein in this tumor progression-related resistance to apoptosis. However, the two variants of highest malignancy, TAU-42 and TAU-46, did not follow the same trend, since they displayed a relatively high content in apoptotic cells and a low Bcl-2 content. Fas receptor expression did not correlate with tendency to apoptosis, indicating that malignant behavior in the AKR lymphoma does not depend on CD95/Fas/APO1 downregulation. Overexpression of p53 was observed only in one of the variants of lowest malignancy.

Biological behavior and cell properties of new AKR lymphoma malignancy variants.

The AKR lymphoma-leukemia is a T lymphocyte neoplasm, most suitable as a model for human T cell malignancies. We have been interested in the process of tumor progression in the AKR lymphoma system. In the present study, two newly isolated variants, the TAU-42 and TAU-44, were characterized with respect to their biological behavior, by comparing them to a previously studied low-malignancy variant, the TAU-39. While the TAU-44 variant formed large s.c. local tumors, the TAU-42 variant formed only small growths or none at all. The TAU-42 lymphoma was found to have the highest malignant potential: it displayed very marked dissemination to spleen, lymph nodes, liver and lungs. The TAU-44 variant had an intermediate degree of metastatic potential but presented a predilection for spread to lymph nodes and spleen and was sometimes found to metastasize to peculiar organs, such as heart and pancreas. Cells derived from the different lymphoma variants varied in their immunophenotype: the highly malignant variant cells expressed more CD4 antigen than the low-malignancy one. The opposite was observed with regard to CD8. The variant cells also differed in their migrating capacity, the more malignant one exhibiting a higher motile activity. Studies on the tumor progression model of AKR lymphoma might contribute to the elucidation of the features determining the aggressiveness of T lymphocytic malignancies.

Comparison of growth rate of two B16 melanomas differing in metastatic potential in young versus middle-aged mice.

The rise of cancer frequency as a function of age is a well-established fact. The aspect of the host age-tumor progression relationship, namely the slower metastatic spread in aged patients, has been investigated to a lesser extent. In the present study, we examined whether host-age-dependent growth rate varies with metastatic capacity of the tumor. The parental B16 and the B16/Col/R, a highly metastatic variant, were employed. A more pronounced growth of both tumors in young as compared to middle-aged mice was found. However, the differential growth in middle-aged versus young mice was more evident in the highly metastatic variant. According to the tumor size data, a sixfold growth reduction in middle-aged mice was observed with B16/Col/R and an only twofold growth reduction was seen with the B16 melanoma. The data might eventually contribute to the finding of more appropriate treatment modalities for the middle-aged cancer patient.

Apoptosis, cell proliferation and in vivo biological behaviour of primary and metastatic tumour cells of an AKR lymphoma variant.

The possibility that apoptosis and/or cell proliferation have a role in tumour progression in a murine T cell lymphoma was tested. The model consisted of the comparison of primary (PT) and metastatic tumour (MT) cells. The PT cells, but not the MT cells displayed a very pronounced tendency for spontaneous apoptosis. Proliferative capacity of MT cells was lower than that of PT cells, suggesting that it does not contribute to the metastatic phenotype in this system. Release from apoptosis does however, probably, play a role in the aggressiveness of the lymphoma.

Metastatic potential and multidrug resistance correlation in the B16 melanoma system.

The question of whether metastatic potential and drug resistance are related phenotypes was addressed by comparing the biological behavior of the parental B16 melanoma and a multidrug resistant variant derived from it, the B16/Col/R. A more pronounced metastatic spread to lungs was observed in mice inoculated i.v. with the B16/Col/R variant than in those bearing the parental line. In addition, in the mice injected with the drug resistant melanoma, unusual tumor masses were observed. Large abdominal and spinal cord growths were seen with the MDR variant but not encountered in mice inoculated with the original B16 melanoma. We further attempted to test the capacity of the two cell types to perform several cellular functions relevant to the metastatic process. The B16/Col/R cells displayed a higher aggregability and cell motility than did the B16 cells. Adherence to endothelial cells was higher in the parental line than in the B16/Col/R, possibly supporting a more efficient extravasation of the variant cells. The drug resistant variant displayed a higher capacity to grow locally in kidney, spleen, cecum and peritoneum, as compared to the parental melanoma, indicating a higher ability of homing and growth in these potential target organs for metastasis. A correlation between metastatic potential and multidrug resistance appears therefore to exist in the system examined.

Cell membrane fluidity and adriamycin retention in a tumor progression model of AKR lymphoma.

Counteraction of drug resistance is a major challenge in cancer therapy, particularly in advanced stages. The main mechanism of multidrug resistance is related to an increased drug efflux. In the present study we examined the effect of modifying cell membrane lipid fluidity on uptake of adriamycin (ADR) in cells of AKR lymphoma malignancy variants. Modification of cell membrane fluidity, either by lecithin or by lecithin-cholesterol mixtures, induced in a high proportion of cells of all variants a higher capacity to accumulate ADR. The chemosensitizing effect, for lecithin in particular, was proportional to the degree of malignancy of the lymphoma variants. The increased ADR uptake was up to 1.4-fold in the variant of lowest malignancy and up to 5-fold in the one of highest aggressiveness. This tendency correlates with our previous studies and is of particular value since highly-malignant tumors are often drug resistant. The cholesterol-lecithin mixture, induced, however, in part of the variants the appearance of a small subpopulation with very low ADR permeability. Cell membrane rigidification is of value for exposing tumor cell cryptic antigens but may be deleterious when used in conjunction with chemotherapy.

Metastasis-related cell functions in primary and metastatic tumor cells of AKR lymphoma.

The metastatic phenotype is of extreme complexity. To complete all the stages of metastasis, the tumor cell must possess a whole series of functional abilities. Multiple biological markers are therefore needed to achieve a deeper understanding of the metastatic phenotype. In the present study we compared primary (PT) to metastatic tumor (MT) cells of two AKR lymphoma variants with respect to several cellular functions relevant to various steps of tumor dissemination. The MT cells of the TAU-44 variant had a higher capacity than the PT cells to attach to endothelial monolayers and ECM, exhibited a more elevated motility and a higher capacity to grow in the spleen as a metastatic target organ. However, the TAU-44-MT cells had a lower ability to grow in the kidney than the PT cells. The TAU-33-MT cells had a higher ability to attach to endothelial cells and to grow in both spleen and kidney but were less motile compared to PT cells. Metastatic cells showed, on the whole, higher ability to perform in most, but not all, stage-specific models than primary tumor cells.

Role of immune response as determinant of tumor progression in function of host age in the B16 melanoma.

Aging constitutes the major cause for the development of most neoplastic diseases. However, tumors in aged people present with a lower degree of aggressiveness than in young patients. The reasons for this paradoxical behavior are not clear. We attempted to verify whether the immune system has a role in the relation between host age, immune response and tumor progression. We compared the growth rate of B16 melanoma and a highly malignant variant, the B16/Col/R, in young and aged mice that have or have not undergone splenectomy. The following results were obtained: (1) Splenectomy stimulated growth in the parental melanoma in both young and aged mice, indicating a protective role of the spleen against this tumor at all ages; (2) Spleen ablation provoked inhibition of the highly-metastatic variant growth in young mice, suggesting a stimulatory role of the spleen in this case; (3) By contrast, in aged mice inoculated with the B16/Col/R variant, splenectomy enhanced tumor growth, indicating a defensive role of the spleen. Age favors a positive host response against the aggressive clone of the melanoma. Differential host response in young versus aged mice can explain, in this tumor system, the difference in tumor progression rate as a function of age.

Comparison of splenectomy effects as an indication for host response to growth of primary and metastatic tumour cells in two murine tumour systems.

The tumour-host relation is of extreme complexity; moreover, it may change during tumour progression. Information regarding this relation may be of importance in appreciating the efficiency of immunotherapy. In the present study, the effect of splenectomy on tumour growth in two murine tumour progression models, the Lewis lung carcinoma and AKR lymphoma, was examined. The effect of spleen ablation on growth of cells derived from primary and metastatic tumour cells was tested. The data obtained showed a differential effect of splenectomy on the growth of primary versus metastatic tumour cells in the two tumour systems, indicating a differential host response induced by the two cell types and/or a differential tumour cell sensitivity to immune reactions. Surprisingly, the spleen appeared to have a defensive role against metastatic tumour cells and a growth enhancing influence on primary tumour cells. The instability of the tumour-host relation may have important implications for the chances of immunotherapy to serve as an efficient cancer treatment.

Correlation between malignancy level of AKR lymphoma variants and sensitivity to hyperthermia.

The tumor progression process has been found to be accompanied by various cell membrane modifications. This cell organelle may therefore be considered as a target for drugs directed against tumor cells of advanced cancer. Hyperthermia acts on tumor cells largely, although not only, via an effect on the cell membrane. In the present study, the in vitro effect of hyperthermia on the tumorigenicity of cells derived from two AKR lymphoma variants of malignancy, TAU-39 of low (LM) and TAU-38 of high-malignancy (HM), was compared. The cells of the HM variant were markedly more sensitive to hyperthermic treatment than those of the LM one. Pretreatment of cells at 41 degrees C or 43 degrees C resulted in a more marked delay in tumor appearance in mice injected with the HM than in those inoculated with the LM variant. Moreover, in mice inoculated with cells pretreated at 45 degrees C, long term survivors were found only in those inoculated with the HM variant. These results corroborate our previous data regarding the effect of hyperthermia on metastatic and primary tumor cells of AKR lymphoma as well as the F1 and F10 variants of B16 melanoma.

Metastasis-associated cell functions in AKR lymphoma malignancy variants.

Multiple structural and functional cell properties are responsible for the complex phenotype of the metastatic cell. Cells derived from AKR lymphoma malignancy variants were compared with regard to several cellular functions relevant to the metastatic behavior. Metastatic behavior correlated with relevant in vitro cell activities, such as cell motility, homotypic and heterotypic adhesion as well as proteolytic activity. The Variant displaying the highest degree of malignancy, TAU-42, exhibited the most elevated ability to perform almost each of the cell functions examined: motility, aggregability, capacity to adhere to endothelium and extracellular matrix, heparanase and protease activity and ability to grow in the spleen. The TAU-33 variant was second in rank in the performance of the above activities. The TAU-44 variant cells, the third in rank of malignancy, displayed some peculiar functional behavior: while manifesting the lowest homotypic adherence and heparanase activity, they had a high predilection for growth in kidney.

Augmentative effects of intracellular chemotherapy penetration combined with hyperthermia in human ovarian cancer cells lines.

A significant proportion of ovarian cancer patients do not achieve a complete response to chemotherapy, due mainly to the evolution of clones resistant to cytotoxic drugs. Exploring possibilities to overcome this resistance constitutes a challenge in the study of ovarian cancer experimental therapy. In the present study, we tested the effect of hyperthermia (40 and 43 degrees C) in combination with adriamycin on three human epithelial ovarian cell lines: OC-109, OC-238, and OC-7-NU. The first was derived from the mucinous and the other two from serous cystadenocarcinomas, and all proved to be tumorigenic in nude mice. FACS analysis showed a pronounced increase in intracellular adriamycin accumulation in the presence of hyperthermia. A significant effect (P < 0.0005) observed at 40 degrees C was even more pronounced at 43 degrees C with the three cell lines. High percentages of cells (up to 70%) shifted into higher fluorescence intensities. The lines differed in their sensitivity to the hyperthermia-induced increase in adriamycin accumulation. Under mild conditions, the OC-109 line was more sensitive than the OC-238 and the OC-7-NU lines. Quantitative determination of intraneoplastic adriamycin by spectrofluorometry also showed a hyperthermia-related increase in intracellular adriamycin (P < 0.005). Our results may indicate that supranormal temperature might serve as an alternative chemosensitizer which lacks the deleterious side effects of other chemosensitizing options.

Enhancing effect of ATP on intracellular adriamycin penetration in human ovarian cancer cell lines.

Ovarian cancer has the highest mortality rate of all gynecological malignancies probably due to the evolution of clones resistant to cytotoxic drugs. Exploring possibilities to overcome such resistance constitutes a challenge in this study. We present the effect of adenosine triphosphate (ATP), serving as a chemosensitizer, in combination with adriamycin on three human ovarian cancer cell lines of epithelial origin, OC-109, OC-238 and OC-7-NU, obtained from malignant ascites of different patients, and were proven to be tumorigenic in nude mice. The three lines differ in their sensitivity to the ATP-induced increase in adriamycin accumulation. FACS analysis showed a pronounced increase in intracellular adriamycin accumulation after treatment with various concentrations of ATP. In the OC-238 line, a 50.1% increase was observed at a low ATP concentration (200 microM), whereas higher concentrations (400 microM and 500 microM) were needed to obtain an increase in ADR accumulation of 30% with the other two lines. Our study demonstrates that ATP improves the penetration of adriamycin at the neoplastic cellular level. Furthermore, our results may indicate that intratumoral ATP may serve as an alternative chemosensitizer which lacks the deleterious side effects of other chemosensitizing options.

Drug resistance and its counteraction by cyclosporin A in function of metastatic potential in the Lewis lung carcinoma system.

Tumor progression (TP) is often accompanied by evolution of drug resistant clones. Decreased intracellular accumulation of cytotoxic agents is probably the major mechanism of drug resistance. In the present study, we tried to examine the possibility to overcome the resistance to adriamycin (ADR) treatment, by cyclosporin A (CS) in two models of TP in the Lewis lung carcinoma (3LL) system. The first model consisted in the comparison of primary tumor cells (3LL-PT) to metastatic cells (3LL-MT) and the second consisted in comparison of lung metastases of the highly malignant variant D122 to those of the parental 3LL tumor. Cyclosporin had a weak augmenting effect on ADR uptake, in the two more malignant cell variants and no influence on the 3LL-PT cells, according to FACS analysis. Cytofluorometry also showed practically no effect of CS on cell size, unlike the effect of other chemosensitizers, such as membrane active agents. In order to find out whether CS counteracts resistance to ADR despite the fact that it does not increase cytotoxic agent uptake, we examined its effect on in vitro proliferative capacity of the 3LL-PT cells. CS in combination with ADR had a more pronounced effect, as compared to single treatments on cell proliferation. The low effect of CS on ADR uptake according to FACS analysis, and by contrast, its efficiency to overcome resistance to ADR according to the in vitro growth results, suggest that the mechanism of the CS action as a chemosensitizer is not related to the p-glycoprotein (P-G-P), known to be overexpressed in the typical multidrug-resistance (MDR) phenotype. A better understanding of the complexity of MDR mechanisms may contribute to the design of new modalities to overcome this phenomenon, which still limits effectiveness of cancer cure, to the early stages of the disease.

Antitumoral activity of an immunomodulatory fraction of Nocardia opaca: mechanism of action.

Immunomodulatory substances have been used as antineoplastic agents in experimental and human systems. Many of these agents were derived from microorganisms. Several biologically active fractions have been isolated from Nocardia. These derivatives were shown to induce interferon production, to activate natural killer cells and macrophages and to exert an antitumoral effect. We attempted to examine the mechanism of the antitumoral activity of the Nocardia water-soluble mitogen (NWSM). The tumor tested was the Lewis lung carcinoma (3LL). Regular histological examination and identification of the cellular immune reaction by monoclonal antibodies against macrophages (Mac 1 antigen), B- (IgG expressing) and T-lymphocytes (anti-Lyt-1), analysed by flow cytometry, were performed on samples of the tumor site and of the spleen. Intratumoral administration of the immunomodulators resulted in a massive accumulation of inflammatory cells around the tumor in mice treated with NWSM. The thick rim of infiltrating cells consisted of macrophages and lymphocytes, while the nontreated tumor was found to provoke only a scanty lymphocyte infiltration. Macrophages were, therefore, present at the tumor site and were directly implicated in the antitumoral effect of the Nocardia immunomodulator. T-lymphocytes were also observed at the site of the tumor. The spleen reaction consisted of marked extramedullary hematopoiesis and enlarged follicles containing prominent germinal centres (assessed also by a FACS-demonstrated increase in B-lymphocytes). In view of the inefficiency of chemotherapy in the treatment of advanced cancer, it is of major importance to explore alternative cancer treatment modalities. Immunotherapy is a particularly interesting alternative since it can potentially affect metastatic disease.