Influence of organ microenvironment on pigmentation of a metastatic murine melanoma

The purpose of these studies was to investigate the relationship of the host microenvironment to the metastatic and pigmented phenotypes of the SW-1 variant of the murine K-1735 melanoma. The SW-1 subline was isolated from an amelanotic lung metastasis in a C3H/HeN mouse given an s.c. injection of the K-1735 melanoma. Cells of this line were highly metastatic and produced tumor deposits in many organs. In all sites except the brain, these lesions were predominantly amelanotic. K-1735 SW-1 cells were isolated from metastases in various organs and subsequently reinoculated into normal syngeneic recipients. Whereas the metastatic phenotype remained stable and thus was heritable, pigmentation was unstable and appeared to be modulated by the site of tumor growth. Further differences in the phenotype of K-1735 SW-1 cells growing in vivo and in culture were revealed by assays for tyrosinase activity. K-1735 SW-1 cells growing in culture did not produce melanin nor did they respond to agents that can stimulate melanin production in another mouse melanoma, the B16 line. K-1735 SW-1 cells do not, however, lack tyrosinase, since these cells are capable of producing melanin when growing in certain organs in vivo. We conclude that the host organ environment may influence a phenotype of malignant melanoma cells, i.e., pigmentation. These findings also suggest caution when extrapolating the results of in vitro biochemical assays to properties of tumor cells growing in vivo.     

Influence of fibroblasts on the invasion and migration of highly or weakly metastatic b16 melanoma cells

We have examined the influence of fibroblasts on the invasive and migratory potential of highly metastatic melanoma B16-BL6 and weakly metastatic B16-FI cells in vitro. Co-culture of B16-BL6 cells with a fibroblast monolayer without cellular contact in a Transwell chamber more effectively induced tumor-cell invasion into Matrigel basement membrane than co-culture of B16-FI cells with a fibroblast monolayer. The activity was closely correlated with the chemotactic migration of tumor cells toward the fibroblast monolayer. We also found that the conditioned medium (CM) from the co-culture of fibroblasts with B16-BL6 cells without cellular contact, i.e., CM (B16-BL6/fibroblast), rather than from co-culture with B16-FI cells, could potentially promote the migration of tumor cells of both types. Tumor cells did not chemotactically migrate to the CM (B16-BL6), CM (B16-FI) or CM (fibroblast). Antibodies against TGF-β1 or FN almost completely abolished the chemotactic migration of B16-BL6 cells to the CM (B16-BL6/fibroblast) or CM (TGF-β1 -treated fibroblast) when these antibodies were c-incubated with fibroblasts and either B16-BL6 or TGF-β1. In contrast, the anti-EGF antibody did not show any inhibitory effects. Analysis of amounts of TGF-β1 or FN in various CM using ELISA plates, and using their specific antibodies, revealed that the concentration of TGF-β1 in the CM (B16-BL6) was slightly higher than in the CM (B16-FI), and the amount of FN in the CM (B16-BL6/fibroblast) was twice as high as in the CM (B16-FI /fibroblast). These results suggest that TGF-β1 released from B16-BL6 cells can stimulate fibroblasts to produce FN; consequently, the tumor cells were able to chemotactically migrate toward the released FN, and the differences in invasive and migratory activities towards fibroblasts in B16-BL6 and B16-FI cells may in part be due to the amounts of TGF-β1 from tumor cells and of FN from TGF-β1 -stimulated fibroblasts.     

Isoenzymes of beta-hexosaminidase from normal rat colon and colonic carcinoma.

N-Acetyl-beta-D-hexosaminidase isolated from normal rat colon was compared to that obtained from a transplantable rat colonic carcinoma. Levels of total hexosaminidase from purified epithelial cells of normal colon were similar to those from purified malignant cells from the transplantable tumor. Cultured malignant cells had significantly higher levels of activity than did freshly purified tumor cells. Isoelectric focusing of hexosaminidase from normal rat colon indicated approximately equal amounts of A (pI 5.0 ) and B (pI 8.1) isoenzyme activity. The B isoenzyme (normal cell) was more stable to heat inactivation than was the A isoenzyme and had significantly higher activity at low pH's. In contrast, the B isoenzyme from the tumor was relatively unstable to heat and low pH.     

Significance of freshly cultured fibroblasts from different tissues in promoting cancer cell growth

The interactions between human cancer cells and primary cultured human fibroblasts without cell-to-cell contact were investigated using double soft-agar culture. Human fibroblasts obtained from different organs were cultured in monolayers and used after the 3rd or 4th passage. In double soft-agar culture, colony formations of cancer cells in the overlayer were stimulated or inhibited by the presence of various kinds of fibroblast in the underlayer. The growth of all cancer cells tested was always stimulated by the presence of fibroblasts obtained from an organ in which cancer cells had already developed, and inhibited by those from skin. However, fibroblast-conditioned media failed to affect cancer cell growth, either in MTT assay or in soft-agar culture. These results suggest that mutual growth reliance exists between human cancer cells and primary cultured fibroblasts by diffusible factors secreted by both cells (paracrine growth) and that mutual growth enhancement occurs between cancer cells and fibroblasts derived from tissues in which cancer cells had originated.     

Heptocyte Growth Factor-Pleiotropic Cytokine Produced by Human Leukemia cells

Hepatocyte growth factor (HGF) was identified. purified and molecularly cloned as a potent mitogen for mature rat hepatocytes in primary culture. It is one of the largest cytokines and is composed of disulfide-linked subunits of approximately 60 (heavy chain) and 35 kilodaltons (light chain). Recent observations revealed that HGF is mitogenic to various epithelial cells other than hepatocytes and to endothelial cells, and that it also acts as a motogen, morphogen and tumor-suppressor as well as a mitogen. These various biological activities of HGF are presumably transduced through the same reccptor, c-Met, which is a member of the tyrosine kinase receptor family. Although it shows multiple biological activities on cells in culture. HGF is most likely the physiological hepatotrophic factor which triggers liver regeneration. It may also function as a renotrophic and pulmotrophic factor after tissue injury. HGF production in the liver. kidney and lung increases after injury to these organs. An elevated HGF level may act as an inducer of compensatory DNA synthesis. The regulation of HGF production is. therefore, important for the control of organ regeneration. HGF is produced mainly by mesenchymal cells such as fibroblasts and vascular smooth musclc cells. Various types of human leukemia cells also secrete HGF both in virru and in riw. Some biological activities of HGF on hematopoietic cells, including co-mitogenic activity on myeloid leukemia cell lines. were recently demonstrated. HGF gene expression and the protein production in leukemia and fibroblast cells are modulated by various cytokines and hormones. Those modulators may indirectly affect organ regeneration and other biological processes by controlling HGF production.     

Direct antiproliferative effects of recombinant human interferon-alpha B/D hybrids on human tumor cell lines

The purpose of these studies was to examine the antiproliferative properties of 16 recombinant human IFN-alpha B/D hybrids against various human tumor lines of different histological origin and to determine whether any of the hybrid molecules possessed immunomodulating activity that could active antitumor properties in peripheral blood monocytes of normal donors. Hybrids with the B domain at the NH2 terminal end exhibited higher activity for antiviral activity and a higher level of direct antitumor antiproliferative activities as compared with hybrids with the D domain at the NH2 terminal end. The positive hybrids were directly cytostatic to melanoma, glioblastoma, renal carcinoma, colon carcinoma, and prostatic carcinoma cells. Tumor cell sensitivity to IFN-alpha hybrids was independent of sensitivity to IFN-gamma or to Adriamycin. The growth of a normal cell line (human embryo fibroblast) was unaffected by IFN-alpha hybrids but was completely arrested by Adriamycin. Some of the IFN-alpha hybrids were also cytostatic to mouse melanoma, lung carcinoma, and fibrosarcoma cell lines, albeit at lower levels than they were to human cells. The incubation of monocytes with IFN-alpha hybrids with the B domain at the NH2 terminal end was also associated with marked antitumor cytotoxicity. Kinetic studies, however, indicated that this activity was attributable to IFN-alpha carried on monocytes and acting directly on tumor cells. We conclude that recombinant human IFN-alpha B/D hybrids possess potent direct antiproliferative activity against a large variety of human tumor lines.     

Preferential growth stimulation of metastatic rat mammary adenocarcinoma cells by organ-derived syngeneic fibroblasts in vitro.

To determine whether organ-derived fibroblasts differentially affect the growth of cells from tumors that preferentially metastasize to specific organs, we investigated the effect of medium conditioned with primary cultured rat fibroblasts from various organs on the in vitro growth of metastatic cell lines and clones of the rat 13762NF mammary adenocarcinoma. The conditioned medium from fibroblasts derived from rat mammary fat pad differentially stimulated tumor cell growth in monolayer culture and clonogenic growth in soft agarose of the highly metastatic clone MTLn3 in a dose-dependent manner. Conditioned medium from fibroblasts derived from the lung and liver also stimulated the growth of clone MTLn3 cells but to a lesser extent than did mammary fat pad fibroblasts. In contrast, poorly metastatic cell clones (MTC, MTPa) did not respond to the growth stimulatory factor(s) from the fibroblast-conditioned medium. The factor(s) responsible for the growth stimulation were inactivated by heat and trypsin treatment and inhibited by low pH and cycloheximide. The result suggest that fibroblasts in different organs have different effects on tumor cell growth, and they may determine, in part, the organ specificity of tumor development and metastasis.     

Inhibition of Tumor Cell Invasion by Ubenimex (Bestatin) in vitro

We have investigated the effect of the immunomodulator ubenimex (bestatin) on tumor cell invasion of reconstituted basement membrane (Matrigel). The invasion of B16-BL6 melanoma cells and Lewis lung carcinoma (3LL) cells into Matrigel-coated filters was inhibited by the presence of bestatin in a concentration-dependent manner. The prctrcatment of either tumor cells or Matrigel with bestatin, however, had little effect on the invasion of tumor cells. Since bestatin was found to inhibit aminopeptidase in addition to its immunomodulating activities, the inhibition of tumor invasion by bestatin is likely to be associated with the action as an enzyme inhibitor. Other aminopeptidase inhibitors, arphamcnine B and amastatin A, could also inhibit tumor cell invasion into Matrigel. Bestatin inhibited hydrolyzing activities towards substrates of aminopeptidases in B16-BL6 melanoma cells. However, bestatin did not have any effect on the haptotactic migration and adhesion of tumor cells to the substrates. These results indicated that bestatin may inhibit tumor cell invasion through a mechanism involving its inhibitory action on aminopeptidases in tumor cells.     

Activation in vitro of mouse macrophages by syngeneic, allogeneic, or xenogeneic lymphocyte supernatants.

Macrophages from normal C57BL/6 mice, those with a subcutaneous B16 melanoma, and mice immunized against the tumor were examined for in vitro cytotoxicity to B16 tumor cells. Macrophages were treated by incubation with supernatants from B16 cells grown either in unmixed cultures or in cultures containing syngeneic, normal, or sensitized allogeneic (A mouse), or xenogeneic (rat) lymphocytes. The various treated and untreated macrophages were then cultured for 5 days with viable B16 cells prelabeled with 125I-5-iodo-2'-deoxyuridine; the cultures were terminated, and the extent of destruction of the B16 target cells was determined from the amounts of radioactivity remaining in adherent tumor cells. Of the untreated macrophages, only those from immunized mice were cytotoxic to the tumor cells; macrophages from normal and tumor-bearing mice became cytotoxic by incubation with supernatants from cultures containing lymphocytes from immunized syngeneic mice, sensitized allogeneic mice, or sensitized rats; and macrophages incubated with supernatants from cultures containing normal nonsensitized allogeneic or xenogeneic lymphocytes showed no cytotoxicity. Thes results suggested that macrophages from tumor-bearing animals are potentially cytotoxic to their syngeneic tumors and can be activated by mediators released from sensitized syngeneic, allogeneic, and/or xenogeneic lymphocytes in vitro.     

Macrophage and lymphocyte potentiation of syngeneic tumor cell and host fibroblast collagenolytic activity in rats

The collagenolytic responses of normal rat skin fibroblasts (NRS-F) and rat mammary MTLn3 tumor-derived fibroblasts (Ln3-F) were examined following exposure to rat macrophage (M phi-CM)- and lymphocyte (LYM-CM)-conditioned culture medium and/or tumor cell-conditioned medium. Alveolar, intratumoral, and peritoneal macrophages were prepared from mammary adenocarcinoma-bearing rats, as were the peritoneal lymphocytes. Incubation of the two fibroblast populations with LYM-CM produced a 10- and 7-fold stimulation of collagenolytic activity by NRS-F and Ln3-F cells, respectively. Similarly, exposure of NRS-F and Ln3-F fibroblasts to peritoneal M phi-CM produced a 7- and 4-fold increase in the expression of collagenolytic activity, respectively. Conditioned medium from MTLn2 tumor cells also stimulated the collagenolytic expression of both fibroblast populations. Incubation of tumor-associated Ln3-F or NRS-F fibroblasts with MTLn2 tumor cell-conditioned medium enhanced fibroblast collagenolytic activity approximately 20 and 17 times, respectively. When M phi-CM and LYM-CM were further "conditioned" by a subsequent incubation with MTLn2 tumor cells, each stimulated the expression of collagenolytic activity by both fibroblast populations and this was especially pronounced (120-fold increase) in the response of Ln3-F to LYM-CM further conditioned by MTLn2 tumor cells. The conditioned media derived from M phi, LYM, and MTLn2 tumor cells with or without trypsin activation contained low levels of interstitial-type collagenolytic activity which made no significant contribution to the collagenolytic activity of the stimulated fibroblasts. Some collagenase inhibitory activity, however, was detected in the M phi-CM, suggesting that the actual stimulation of collagenolysis by host fibroblasts is underestimated. We conclude that macrophages, lymphocytes, and tumor cells all have the potential to produce stimulatory factor(s) which enhance the collagenolytic activity of normal fibroblast populations. This study provides further evidence of the multifactorial control of collagenase production and supports the concept that host cell-tumor cell interactions can enhance the expression of collagenolytic enzymes.     

Early tumor growth in metastatic organs influenced by the microenvironment is an important factor which provides organ specificity of colon cancer metastasis

We have previously demonstrated that liver metastases in nude mice and lung metastases in nude rats occurred specifically, when KM12SM human colon carcinoma cells were inoculated orthotopically into the cecal wall of nude mice and rats. To clarify the relationship between the tumor growth potential in the metastatic organs and the metastatic organ preference in these two metastatic models, we have evaluated the in vitro cell growth activities affected by the organ conditioned medium (CM) from the liver and lung, and the in vivo growth activities of the ectopic implanted tumors in the liver and lung. The tumorigenicity of the ectopic implanted tumors was 100% in mouse liver, 33% in rat liver, 50% in mouse lung, and 75% in rat lung. The crude liver CM of the animals showed inhibitory activities for KM12SM cell growth in a dosage-dependent manner, and the crude lung CM stimulated KM12SM cell growth. The liver CM of nude mice inhibited the KM12SM cell growth more strongly compared with the CM of nude rats, and the lung CM of nude rats was more strongly stimulated compared with the CM of nude mice. The liver CM of nude mice had non-heparin binding factors, which stimulated or inhibited KM12SM cell growth, in a molecular weight range of 50 to 100 kDa. By contrast, the liver CM of nude rats showed no growth stimulating activity for KM12SM cells. These results suggest that the metastatic organ specificity of KM12SM cells may depend on the early tumor growth influenced by the microenvironment in metastatic organs.     

Expression of heparanase by platelets and circulating cells of the immune system: possible involvement in diapedesis and extravasation.

Interaction of T and B lymphocytes, platelets, granulocytes, macrophages and mast cells with the subendothelial extracellular matrix (ECM) is associated with degradation of heparan sulfate (HS) by a specific endoglycosidase (heparanase) activity. The enzyme is released from intracellular compartments (i.e., lysosomes, specific granules) in response to various activation signals (i.e., thrombin, calcium ionophore, immune complexes, antigens, mitogens), suggesting its regulated involvement in inflammation and cellular immunity. In contrast, various tumor cells appear to express and secrete heparanase in a constitutive manner, in correlation with their metastatic potential. Heparanase enzymes produced by different cell types may exhibit different molecular properties and substrate cleavage specificities. The platelet enzyme appears also in a latent form. It can be activated by tumor cells and thereby facilitate their extravasation in the process of metastasis. Degradation of ECM-HS by all cell types was facilitated by a proteolytic activity residing in the ECM and/or expressed by the invading cells. This proteolytic activity produced a more accessible substrate for the heparanase enzymes. Heparanase-inhibiting, nonanticoagulant species of heparin markedly reduced the incidence of lung metastasis in experimental animals. These species of heparin also significantly impaired the traffic of T lymphocytes and suppressed cellular immune reactivity and experimental autoimmune diseases. Heparanase activity expressed by intact cells (i.e., platelets, mast cells, neutrophils, lymphoma cells) was found to release active HS-bound basic fibroblast growth factor from ECM and basement membranes. Heparanase may thus elicit an indirect neovascular response in processes such as wound repair, inflammation and tumor development. The significant anticancerous effect of heparanase-inhibiting molecules may therefore be attributed to their potential inhibition of both tumor invasion and angiogenesis. Both normal leukocytic cells and metastatic tumor cells can enter the bloodstream, travel to distant sites and extravasate to the parenchyma at these sites. We suggest that heparanase is utilized for this purpose by both types of cells. Other functions (i.e., enzyme activities, adhesive interactions, chemotactic and proliferative responses) of metastatic tumor cells seem to mimic the equivalent functions of leukocytes as they migrate across blood vessels to gain access to sites of inflammation.     

Enhanced induction of tissue-type plasminogen activator in normal human cells compared to cancer-prone cells following ionizing radiation.

Normal human fibroblast (i.e., GM2936B, GM2907A, and IMR-90) and cancer-prone human fibroblast (i.e., Fanconi's anemia, Bloom's syndrome, and Ataxia telangiectasia) cells demonstrated the induction of intracellular and extracellular levels of tissue-type plasminogen activator (t-PA) at 6 and 12 hr, respectively, following ionizing radiation. Induced t-PA enzymatic activities following ionizing radiation were blocked by actinomycin D treatments. t-PA enzymatic activities were induced over 14-fold in Ataxia telangiectasia cells, over 9-fold in Bloom's syndrome cells, and over 6-fold in Fanconi's anemia cells, as compared to normal human fibroblasts. Similarly, the induction of t-PA mRNA levels in cancer-prone cells were between 5- to 10-fold higher than those observed in normal cells following equitoxic doses of ionizing radiation. Temporal induction of t-PA mRNA levels for normal and cancer-prone human cells were consistent with quantifiable enzymatic activities. The elevated induction of an intracellular protease (i.e., t-PA) in cancer-prone human cells is reminiscent of an "SOS"-like response observed in yeast and bacteria.     

Opposite effects of modifiers of the ApcMin mutation in intestine and mammary gland

Patterns of tumor susceptibility in different organs are widely divergent in mouse strains: one strain may be highly susceptible to tumors in one organ but resistant in another organ, whereas another strain may exhibit the opposite pattern (P. Demant, Semin. Cancer Biol., 3: 159-166, 1992). Therefore, susceptibility to tumors in different organs is assumed to be controlled by different sets of genes. On the other hand, many oncogenes and tumor suppressor genes are mutated in tumors from different organs, indicating that similar tumorigenic pathways operate in various tissues. To obtain insight into the interactions of susceptibility genes with one of such pathways, we compared tumorigenesis in intestine and mammary gland in recombinant congenic strains (RCSs) carrying the Apc(Min) mutation, affecting the Wnt pathway. The presence of Apc(Min) increased considerably the incidence of intestinal and mammary tumors. The individual RCSs differed in the number and latency of Apc(Min)-induced intestinal and mammary tumors and histological type of the latter. Unexpectedly, the strain distribution of susceptibility to the intestinal and mammary tumors in the Apc(Min)-bearing mice was opposite in the RCSs; the strains most susceptible for intestinal tumors were most resistant to mammary tumors and vice versa. This suggests that a set of genes controls the impact of the Apc(Min) mutation in both organs but with opposite effects. Elucidation of the basis of the observed strain differences in organ-specific Wnt pathway-mediated tumorigenesis will help to understand the interactions between germ-line encoded allelic differences in susceptibility genes and the spectrum of somatic mutations in tumor cells.     

Immunohistochemical and biochemical study of a cathepsin B-like proteinase in human colonic cancers

An immunohistological study was carried out on 51 human colorectal adenocarcinomas and eight samples of histologically normal colonic mucosa removed far from tumors, using anti-rabbit cathepsin B and anti-human cathepsin B immunoglobulins. Positive reactions were obtained on tumor cells and macrophage-like cells. However, as these immunoglobulins could not discriminate between cathepsin B and cathepsin B-like proteinases, and as they cross-reacted with cathepsins H and L, a partial characterization of the proteinase activities was performed in order to identify the type of enzyme present in the positive cells. The levels of cathepsins H and L were very low in extracts of colorectal tumors and normal colonic mucosa. A peculiar cathepsin B-like proteinase activity with pH optimum at 6.8 was found in tumor extracts together with the lysosomal cathepsin B, whereas normal colonic mucosa showed only cathepsin B activity (pH optimum, 6.0). These results indicate that lysosomal cathepsin B is responsible for staining of macrophage-like cells found in the lamina propria of colonic mucosa and in the peritumoral stroma. Immunohistochemical staining of colonic tumor cells observed in 29/51 cases seems on the other hand to be primarily due to a cathepsin B-like proteinase. Three colonic tumor cell lines, Colo-205, HT-29, and SW-1116, were also studied using the same methods. These cells produced a latent cathepsin B-like proteinase which, after activation, was similar to that found in tumor extracts. This latent proteinase was detected mainly in the culture media. The cultured colonic tumor cells, after staining by anti-cathepsin B antibodies, showed strongly positive granules. In conclusion, this work demonstrates that malignant colonic cells are the source of a cathepsin B-like proteinase, with optimal activity near neutrality. Its secretion into the extracellular space indicates furthermore, that it may be an important component of the "proteinase cascade" associated with tumor invasion and metastasis.     

Effects of presensitization in vivo on cell-mediated responses to embryonic antigens in vitro

Lymphoid cells specifically reactive with antigens shared by rat bowel carcinomas and mid-term embryo cells were generated by in vitro culture on monolayers of embryo cells. Spleen cells from WF females were cultured for 5 days on monolayers of syngeneic embryo cells or adult cells and assayed for cytotoxic activity on syngeneic embryo, bowel carcinoma, or adult fibroblast target cells in microcytotoxicity and 51Cr-release assays. After culture on embryo monolayers, spleen cells were cytotoxic for embryo and tumor but not for adult fibroblast target cells. Enhanced cytotoxicity was recorded when the spleen cells were cultured from females after interstrain (WF X BN) pregnancy rather than from virgin females. In contrast, previous intrastrain (WF X WF) pregnancy appeared to depress the generation of spleen cells cytotoxic for target cells bearing embryonic antigens.     

Interleukin-4 and breast cancer

IL-4 is a pleiotropic cytokine produced by T lymphocytes which acts on various cells of such as T and B lymphocytes, monocytes, fibroblast, endothelial cells, macrophages and some others. IL-4 was originally described as a B cell growth factor, and now known to provide potent anti-tumor activity against various tumors, including breast cancer. IL-4 can induce apoptosis in cultured breast cancer cells. In addition, it has been clarified that IL-4 plays an important role in the regulation of estrogen synthesis enzymes including 17beta-HSD and 3beta-HSD. These findings imply that IL-4 is a key enzyme not only for Th2 type immune reactions but also for tumor cell growth itself in human breast cancer.     

Interspecies differences in membrane-associated protease activities of thyrocytes and their relevance for thyroid cancer studies

Background

To understand the role of proteases involved in human thyroid cancer progression and tissue invasion, thyrocytes from other species could potentially be used provided their characteristics are similar. It is not known whether dipeptidyl peptidase IV and aminopeptidase N activities, which are overexpressed in human thyroid cancer, are, as in human, also absent in normal thyrocytes of other species, making them suitable models for studies on the regulation of these proteases.

Methods

To assess the role of these proteases, activity was measured in thyroid tissue of human, mouse, rat, porcine, bovine and ovine origin. The lysosomal protease, dipeptidyl peptidase II, was used for comparison.

Results

Murine, rat, ovine, bovine and human thyrocytes all lacked dipeptidyl peptidase IV and aminopeptidase N activity, but porcine thyrocytes were found to possess both. In contrast, lysosomal dipeptidyl peptidase II was strongly expressed in all species. These activity patterns were maintained in cultured cells. Cultured porcine thyrocytes formed follicles with typical morphology upon stimulation with TSH but differed from human thyrocytes in their response to thiamazole.

Conclusions

These species differences in the expression of dipeptidyl peptidase IV and aminopeptidase N, indicate that porcine thyrocytes cannot be considered appropriate for the study of proteases in human cancer development.     

Cyclic nucleotide phosphodiesterase activity in normal and neoplastic rat mammary cells grown in monolayer culture.

The activity of cyclic 3':5'-nucleotide phosphodiesterase (PDE) (EC 3.1.4.17) was measured in cultured normal and neoplastic rat mammary epithelium. Total PDE activity in normal cells was 1.6 to 6 times higher than that in tumor cells over a concentration range of 0.01 to 1 mM cyclic adenosine 3':5'-monophosphate. PDE activity was distributed between the low-speed (4000 x g) particulate and supernatant fractions in both cell lines, with the particulate fraction possessing 60 to 70% of the total. Double reciprocal kinetic plots were nonlinear, suggesting the presence of high- and low-affinity PDE activities. Similar, but not identical biphasic curves obtained from both normal and neoplastic cells suggested that at least two different PDE activities were present in a membrane-bound as well as a soluble form. Apparent Michealis constants for the high-affinity enzyme ranged from 2 to 6 muM; the low-affinity enzyme was 1 mM. In the presence of 10 mM caffeine and at a substrate concentration of 1 muM, PDE activity was inhibited 40 and 80% of basal levels in normal and tumor cells, respectively. In general, the membrane-bound enzyme was inhibited to a greater extent than the soluble, regardless of the cell line examined. Although normal cells exhibited higher PDE activities in terms of total specific activity, when soluble activities were compared at low substrate concentrations, the opposite was the case. At a substrate concentration of 0.01 muM, normal cell, low-Km soluble specific activity was 40% less than comparable tumor cell activity. Our results support the contention that PDE is induced by its own substrate, cyclic adenosine 3':5'-monophosphate. In addition, they suggest that the low cyclic adenosine 3':5'-monophosphate steady-state levels characteristic of malignant cells are maintained by a soluble high-affinity isozyme of PDE.