Selection of liver-colonizing tumor cells from a murine fibrosarcoma induced by methylcholanthrene

Summary An experimental tumor model was developed to study the organ preference of malignant tumors. The primary tumor ER 15-P was induced by in-oculation of 1 mg methylcholanthrene in 0.1 ml sesame oil into the left femoral muscle of a female C57/B1₆J mouse. The tumor was palpable 100 days after induction. Spontaneous lung metastases were found at autopsy on day 128. Serial IM transplantation of tumor cells from the primary ER 15-P resulted in pulmonary metastases in all male and female mice. After IV injection of tumor cells from ER 15-P to male mice, colonies were found in lungs, thoracic cavity, liver, kidneys and occasionally also adrenals; female mice sometimes had ovarian metastases in addition, but no hepatic metastases. Liver-colonizing tumor cells were selected in male mice as follows: (a) IV injection of tumor cells from primary ER 15-P; (b) removal of tumor cells from liver tumor nodules, reinjection into mesenteric vein; (c) preparation of resulting tumors in the liver, reinjection of these cells through the portal system in one group of mice, and IV administration into tail vein in another group: (d) IM inoculation of tumor cells of the mesenteric passage in the left gastrocnemius muscle of mice prior to IV injection via tail vein in another group. Steps c and d were repeated three times. The procedure resulted in a highly significant decrease of tumor cell colonization to lungs and other organs, and a preferential increase of liver colonization. The liver preference of cell lines thus selected was obvious. Possible mechanisms for the organ preference of malignant tumors are discussed.     

In vivo metabolic effects of hyperglycemia in murine radiation-induced fibrosarcoma: a 31P NMR investigation.

The hyperglycemia-induced in vivo metabolic changes produced in subcutaneous murine RIF-1 tumors, grown on female C3H/Anf mice, were examined with 31P surface-coil NMR. Serum glucose levels were elevated 4-fold by bolus intraperitoneal injection of 0.3 ml of an aqueous 50% glucose solution. Tumor pH was calculated from the chemical shift of Pi and relative phosphocreatine and ATP concentrations were determined by Simpson's rule integration of the peak areas. Tumor pH decreased by ca. 0.45 unit over 2 hr while phosphocreatine concentrations decreased by ca. 50% over the same time period (n = 9). Initial tumor pH correlated inversely with the initial peak intensity ratio of Pi:ATP (r = -0.77). In a significant number of tumors (n = 4), two pH populations were observed. In these tumors, one population was unaffected by hyperglycemia and the other showed a decrease in pH. In the other tumors (n = 5), the pH distribution broadened as the pH decreased. In these tumors, the observed decreased in phosphocreatine concentration correlated with that calculated from the effect of measured tumor pH on the intracellular creatine kinase equilibrium (n = 18, r = 0.91). This correlation and consideration of the Pi distribution in the tumor suggest that the pH measured by 31P NMR is weighted heavily by intracellular pH for the RIF-1 tumor. The presence of two distinct tumor pH populations or a broadened pH distribution likely reflects variations in tumor microcellular environment. Control experiments showed negligible changes in tumor pH and high energy phosphate concentrations after bolus intraperitoneal injection of 0.3 ml of isotonic saline. In addition, negligible changes in leg muscle pH and high energy phosphate concentrations were observed after glucose injection into mice with or without tumors. These results indicate that hyperglycemia induced by intraperitoneal glucose injection is effective in lowering the tumor pH of the murine RIF-1 tumor.     

Alteration of brain catecholamines during growth of benzo(a)pyrene induced murine fibrosarcoma.

Brain catecholamines (CA) were studied in discrete brain areas of benzo(a)pyrene (b(a)p) induced fibrosarcoma bearing mice. Dopamine (DA) and norepinephrine (NE) levels decreased significantly in different brain areas especially in corpus striatum and hypothalamus with the tumor progression, indicating an inverse relationship between brain DA and NE levels and tumor growth. Since impaired hormonal and immunological functions are manifestation of systemic alteration during tumor growth, it appears that during malignant growth an alteration of these brain CA may play an important role in the regulation of systemic alterations.     

Role of a tumor-suppressor gene in the negative control of anchorage-independent growth of Syrian hamster cells.

Tumor-suppressor genes control the neoplastic phenotype of tumor cells, but the function of these genes in normal cells is unknown. In this report we show that the loss of a tumor-suppressor gene function releases negative controls on the growth of cells in agar. This conclusion is based on observations of cell hybrids and studies of cell variants that have retained or lost a tumor-suppressor gene function. Nontumorigenic cell hybrids between normal Syrian hamster embryo cells and a benzo[a]pyrene-transformed tumor-cell line (BP6T) continued to secrete autocrine and/or paracrine growth factors produced by the tumor cells but failed to respond to these factors by growing in agar. Normal diploid cells also failed to grow in agar in response to the growth factors produced by the tumor cells. Clonal variants of nontumorigenic, immortal Syrian hamster cell lines were isolated that either retained (termed supB+) or had lost (termed supB-) the ability to suppress tumorigenicity of BP6T tumor cells after cell hybridization. Neither supB+ nor supB- variants grew in agar under conditions that allowed efficient growth of the tumor cells. However, supB- cells were reversibly induced to grow in agar with high colony-forming efficiencies in the presence of tumor cell-conditioned medium or by supplementation of the medium with a combination of growth factors. Under the same conditions, the supB+ cells failed to grow in agar. This enhanced growth-factor responsiveness in agar was used to select for supB- variants existing at a low frequency in the supB+ population. These two phenotypes, loss of tumor-suppressor function and enhanced growth-factor responsiveness in agar, were seen to cosegregate. These results indicate the tumor-suppressor gene function in these cells negatively regulates the growth response of cells in agar to mitogenic stimuli. This growth regulation may depend on cell shape or adhesion because supB+ and supB- cells grown attached to plastic responded similarly to growth factors.     

Pasteurella multocida toxin is a potent inducer of anchorage-independent cell growth.

The growth of many normal cells requires contact with an adhesive substratum, a requirement that is frequently abrogated in the transformed phenotype. We have explored pathways that can lead to the anchorage-independent growth of cultured Rat-1 fibroblasts. Pasteurella multocida toxin (PMT), a 146-kDa mitogenic protein, caused a striking increase in the formation of colonies (greater than 200 microns) from single cells in soft agar. The magnitude of the effect of PMT was greater than that achieved by epidermal growth factor or platelet-derived growth factor. The toxin was extremely potent, with half-maximal and maximal effects observed at 1 and 10 pM PMT, respectively. This concentration dependence of the action of the toxin is similar to that for the stimulation of DNA synthesis in adherent cultures of the cells. Stimulation of colony formation could be achieved by a transient exposure of the cells to PMT and it was blocked by methylamine, indicating that the toxin enters the cells to act. Colony formation was stimulated equally by native and recombinant PMT, but a truncated version (33.5 kDa) of the recombinant toxin was ineffective. PMT antiserum blocked colony formation in response to PMT. In the Rat-1 cells, PMT stimulated the phospholipase C-mediated hydrolysis of inositolphospholipids, as indicated by the stimulation of inositol phosphate release, Ca2+ mobilization, and phosphorylation of a protein kinase C substrate. The results indicate that the deregulation of signal-transduction pathways as elicited by an intracellularly acting bacterial toxin can induce a malignant phenotype.     

Endothelial cells from bovine adrenal medulla develop capillary-like growth patterns in culture.

The endocrine barrier between chromaffin cells and the blood stream in the adrenal medulla is made of capillary endothelial cells. We have now succeeded in isolating endothelial cells from adrenal medullary tissue, which are probably derived from this barrier. These cells grow on plastic surfaces in the absence of special growth factors or collagen overlays and differentiate into organized structures quite similar to true capillaries. The cells contain factor VIII:R, a marker for endothelial cells, and form intercellular junctions characteristic of capillary endothelial cells. They also synthesize and secrete basal lamina structures and engage in transcytosis, a characteristic ultrastructural and functional combination of exocytosis and endocytosis across the thin endothelial cell processes. These endothelial cells can take up and deaminate catecholamines by A-type monoamine oxidase, an enzyme functionally distinct from the B-type monoamine oxidase found in chromaffin cells. These data indicate that the chromaffin cell and its endothelial cell neighbor may constitute the functional unit of catecholamine metabolism in the adrenal medulla.     

Somatostatin suppresses growth of murine myeloid leukemia in vivo

Daily injections of somatostatin into mice with myeloid leukemia retarded the tumor growth. This myeloid leukemia is an insulin- dependent tumor (in that it grows more slowly in hypoinsulinemic diabetic mice than in nondiabetic animals). Since somatostatin decreased the level of immunoreactive insulin in mice with myeloid leukemia, and since the treatment with insulin abrogated the antileukemic effect of somatostatin, we attribute retarded growth of this leukemia to decreased secretion of insulin, caused by somatostatin.     

In vivo selection of wild-type hematopoietic stem cells in a murine model of Fanconi anemia.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by birth defects, increased incidence of malignancy, and progressive bone marrow failure. Bone marrow transplantation is therapeutic and, therefore, FA is a candidate disease for hematopoietic gene therapy. The frequent finding of somatic mosaicism in blood of FA patients has raised the question of whether wild-type bone marrow may have a selective growth advantage. To test this hypothesis, a cohort radio-ablated wild-type mice were transplanted with a 1:1 mixture of FA group C knockout (FACKO) and wild-type bone marrow. Analysis of peripheral blood at 1 month posttransplantation showed only a moderate advantage for wild-type cells, but upon serial transplantation, clear selection was observed. Next, a cohort of FACKO mice received a transplant of wild-type marrow cells without prior radio-ablation. No wild-type cells were detected in peripheral blood after transplantation, but a single injection of mitomycin C (MMC) resulted in an increase to greater than 25% of wild-type DNA. Serial transplantation showed that the selection occurred at the level of hematopoietic stem cells. No systemic side effects were observed. Our results show that in vivo selection for wild-type hematopoietic stem cells occurs in FA and that it is enhanced by MMC administration.     

Regulation of microfilament organization and anchorage-independent growth by tropomyosin 1.

Variants of chemically immortalized Syrian hamster embryo cells that had either retained (supB+) or lost (supB-) the ability to suppress tumorigenicity when hybridized with a fibrosarcoma cell line were subcloned. Both supB cell types are nontumorigenic; however, the supB- but not supB+ cells exhibit conditional anchorage-independent growth. Alterations of actin microfilament organization were observed in supB- but not supB+ cells that corresponded to a significant reduction of the actin-binding protein tropomyosin 1 (TM-1) in subB- cells. To examine the possibility of a direct relationship between TM-1 expression and the subB- phenotype, subB+ cells were transfected with an expression vector containing the TM-1 cDNA in an antisense orientation. The antisense-induced reduction of TM-1 levels in supB+ clones caused a microfilament reorganization and conferred anchorage-independent growth potential that were indistinguishable from those characteristic of supB- cells. These data provide direct evidence that TM-1 regulates both microfilament organization and anchorage-independent growth and suggest that microfilament alterations are sufficient for anchorage-independent growth.     

Hexamethylenebisacetamide-resistant murine erythroleukemia cells have altered patterns of inducer-mediated chromatin changes.

We determined inducer-mediated changes in chromatin structure near the globin genes in a variant line of murine erythroleukemia cells (MELC). The variant cell line, R1, was derived from the inducer-sensitive DS19 cell line by selection for inducer-resistance. R1 cells are resistant to induction of erythroid differentiation by hexamethylenebisacetamide (HMBA) whereas the parental line is HMBA-sensitive. Uninduced MELC (both inducer-sensitive DS19 cells and inducer-resistant R1 cells) have DNase I-sensitive sites in chromatin containing the alpha 1- and beta maj-globin genes. These nuclease-sensitive regions are located within the beta maj-globin second intervening sequence (IVS2) and near the alpha 1-globin gene 5' cap site. Culture with HMBA causes changes in chromatin structure in both parental and variant cell lines. In DS19 cells, the DNase I-sensitive site within the beta maj-globin IVS2 becomes more resistant to nuclease cleavage, and a new DNase I-sensitive region develops near the beta maj-globin cap site. In addition, the nuclease-sensitive region adjacent to the cap site of the alpha 1-globin gene increases, and a novel 5' nuclease-sensitive site is also established. In R1 cells, HMBA-mediated changes in chromatin structure are incomplete. The DNase I-sensitive site within the beta maj-globin IVS2 becomes more resistant to nuclease cleavage, but the nuclease sensitivity near the beta maj-globin cap site does not increase to the extent observed in DS19 cells. The pattern of nuclease sensitivity near the alpha 1-globin gene is essentially unchanged after culture of R1 cells with HMBA. Thus, in R1 cells, resistance to HMBA-induced expression of globin genes is associated with failure to detect inducer-mediated changes in chromatin structure 5' to the cap site of the alpha 1- and beta maj-globin genes. These results also suggest that the increased nuclease resistance of a site in the beta maj-globin IVS2 does not depend on the establishment of a DNase I-sensitive region near the beta maj-globin gene cap site.     

Adoptive transfer of immunity against solid fibrosarcoma in mice with splenocytes and peritoneal exudate cells obtained after in vitro sensitization and in vivo immunization with cis-dichlorodiamine platinum(II) treated fibrosarcoma cells

Co-cultivation of splenocytes with cis-dichlorodiamine platinum (II) treated tumor cells generate cytotoxic splenocytes, which when injected into normal mice, render them resistant to tumor challenge. Significant increases in mean survival time and 33% of tumor free survivals were observed in mice exposed to a tumor challenge on the 10th day after injection of sensitized splenocytes. Splenocytes peritoneal exudate cells obtained after in vivo immunization of mice with cis-dichlorodiamine platinum(II) treated cells retarded tumor growth in vivo when injected in different combinations in tumor bearing mice. Maximum survival time of tumor bearing mice and 20% tumor free survivals were observed when the animals were injected with a combination of immune splenocytes and normal peritoneal exudate cells. The increase in the number of macrophages of immunotherapeutically treated mice suggests that host macrophages have been activated. Splenocytes and macrophages obtained from immunotherapeutically treated mice showed an increase in cytotoxicity against tumor cells in vitro.     

Corticosteroid effect on murine hemopoietic precursor cells in vivo

The effect of methylprednisolone on murine hemopoietic colony formation in diffusion chambers implanted in mice was evaluated. A dose-dependent increase in granulocytic colony (CFU-DG) formation from murine marrow was observed. This effect could be abrogated by administration of progesterone. These studies suggest that the murine early granulocytic precursors (CFU-DG) have receptors that mediate proliferation-promoting signals triggered by glucocorticoids. Erythroid colony formation (CFU- DE) was not effected by methylprednisolone administration.     

Leukotriene C: a slow-reacting substance from murine mastocytoma cells.

Murine mastocytoma cells treated with calcium ionophore A23187 produced a slow-reacting substance (SRS) that caused guinea pig ileum to contract. The response was reversed by the SRS antagonist FPL 55712. On the basis of isotope incorporation experiments, spectroscopy, and chemical degradations, the SRS was identified as a cysteine-containing derivative of 5-hydroxy-7,9,11,14-icosatetraenoic acid. This amino acid was attached in thioether linkage at C-6. The SRS is structurally related to previously identified epoxy and dihydroxy metabolites of arachidonic acid in leukocytes. A common feature is the presence of a conjugated triene, and the name "leukotriene" has been introduced to designate these compounds. Leukotriene A (5,6-epoxy-7,9,11,14-icosatetraenoic acid) is an intermediate in the formation of leukotriene B (5,12-dihydroxy-6,8,10,14-icosatetraenoic acid) and is proposed to be a precursor also of leukotriene C, which is the SRS identified here.