Experimental metastasis of oncogene-transformed NIH 3T3 cells in chick embryo.

By means of a highly sensitive and quantitative assay for specific detection of metastasized tumor cells in chick embryonic organs using the polymerase chain reaction (PCR), we have examined the experimental metastatic ability of individual clones of NIH 3T3 cells, transformed with oncogenes: v-Ki-ras, v-Ha-ras, v-src, v-fos, and v-abl. Such a transformed clone had different metastatic abilities in different embryonic organs. Among them, two clones of NIH 3T3 cells transformed with ras-oncogenes (v-Ki-ras or v-Ha-ras) metastasized to liver and lungs of chick embryo, and grew there more rapidly than the other clones. The parental NIH 3T3 cells were detected as slight bands of PCR products after iv injection, indicating some cells were trapped in chick embryonic organs, but did not grow. These findings indicate that the transformed cells are able to invade the organ tissues and grow in embryonic chick organs, but non-metastatic cells such as the untransformed-NIH 3T3 cells are not able to grow in the secondary sites. These experiments clearly demonstrate the usefulness of this assay system to study genes involved in malignant transformation.     

Antitumor activity of a novel nucleoside, 2'-C-cyano-2'-deoxy-1-beta-D-arabinofuranosylcytosine (CNDAC) against murine and human tumors.

The antitumor effects of 2'-C-cyano-2'-deoxy-1-beta-D- arabinofuranosylcytosine (CN-DAC), a synthetic 1-beta-D-arabinofuranosyl-cytosine (ara-C) derivative, were examined and compared with that of ara-C in murine tumors and in various human tumors using three different chemosensitivity tests. CNDAC extended the life span of mice bearing P388 leukemia. CNDAC had a unique in vitro antitumor spectrum for human cancers different from that of ara-C. Compared with ara-C, CNDAC was more effective in 10 human tumors (2 lung, 4 stomach and 4 osteosarcoma), equal in 2 tumors (lung and fibrosarcoma) and less potent in 11 tumors (4 lung, 4 osteosarcoma, bladder, renal and epidermoid). Characteristically CNDAC showed excellent activities against tumors, refractory to ara-C, such as HT-1080 human fibrosarcoma implanted in chick embryos or athymic mice, although its cytotoxicity against HT-1080 was almost equal to that of ara-C. Thus, CNDAC is an interesting and promising agent that should be considered for further detailed preclinical evaluation.     

Differential effects of polyunsaturated fatty acids on chemosensitivity of NIH3T3 cells and its transformants

Polyunsaturated fatty acids (PUFAs) have been suggested, on the basis of animal-model studies, to be related not only to cancer development but also to chemotherapeutic effects. Controversy persists, however, as to which types of PUFAs are beneficial in terms of chemosensitivity. In this study, we used the NIH3T3 cell line and its SIC(sigmoid colon cancer)-oncogene transformants to investigate the effects of PUFAs on the chemosensitivity of non-malignant and malignant cells in terms of cell proliferation. We also determined the fatty-acid composition of cells by high-performance liquid chromatography (HPLC). The results revealed that the sensitivity of SIC transformants to mitomycin C (MC) was lower than that of NIH3T3 cells cultured in 10% calf-serum DMEM without PUFA supplementation. When cells were cultured in DMEM supplemented with eicosapentaenoic acid (EPA) at a concentration (2 μg/ml) that does not influence cell proliferation, the sensitivity of SIC transformants to MC increased, whereas that of NIH3T3 cells decreased in comparison with the sensitivity of cells cultured without PUFA supplementation (p < 0.05). There was no difference between the 2 cell lines in the chemosensitivity of cells cultured in medium supplemented with arachidonic acid (ARA). The SIC transformants contained more stearic acid (C:18) and less lauric acid (C:12) than NIH3T3 cells cultured without PUFA. Culturing the cells in medium supplemented with EPA or ARA modified the cellular fatty-acid composition. EPA caused the relative combined percentage of lauric acid and myristic acid (C:14) in SIC transformants to decrease significantly, and the SIC transformants tended to accumulate additional EPA, in contrast to the NIH3T3 cells. We conclude that the alterations in fatty-acid composition in malignant transformants caused by exogenous EPA differ from those in non-malignant cells, and that these changes account for the increased chemosensitivity of malignant transformants. Although preliminary, these findings imply that EPA specifically enhances the chemosensitivity of malignant cells. Int. J. Cancer, 70:357–361, 1997. © 1997 Wiley-Liss, Inc.     

Polyamine metabolism and interconversion in NIH 3T3 and ras-transfected NIH 3T3 cells

The effect of transfection of NIH 3T3 cells by the human ras (c-Ha-ras-1) oncogene on uptake, interconversion, and excretion of polyamines was studied. Uptake and interconversion of spermidine were higher in the ras-transfected cells. Acetylpolyamines were excreted into the medium by the ras-transfected cells, whereas they were retained by NIH 3T3 cells. In addition to acetylpolyamines, some unknown polyamine conjugates occurred in the ras-transfected cells.     

Molecular basis for G2 arrest induced by 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine and consequences of checkpoint abrogation

2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) is a nucleoside analogue with a novel mechanism of action that is currently being evaluated in clinical trials. Incorporation of CNDAC triphosphate into DNA and extension during replication leads to single-strand breaks directly caused by beta-elimination. These breaks, or the lesions that arise from further processing, cause cells to arrest in G2. The purpose of this investigation was to define the molecular basis for G2 checkpoint activation and to delineate the sequelae of its abrogation. Cell lines derived from diverse human tissues underwent G2 arrest after CNDAC treatment, suggesting a common mechanism of response to the damage created. CNDAC-induced G2 arrest was instituted by activation of the Chk1-Cdc25C-Cdk1/cyclin B checkpoint pathway. Neither Chk2, p38, nor p53 was required for checkpoint activation. Inhibition of Chk1 kinase with 7-hydroxystaurosporine (UCN-01) abrogated the checkpoint pathway as indicated by dephosphorylation of checkpoint proteins and progression of cells through mitosis and into G1. Cell death was first evident in hematologic cell lines after G1 entry. As indicated by histone H2AX phosphorylation, DNA damage initiated by CNDAC incorporation was transformed into double-strand breaks when ML-1 cells arrested in G2. Some breaks were manifested as chromosomal aberrations when the G2 checkpoint of CNDAC-arrested cells was abrogated by UCN-01 but also in a minor population of cells that escaped to mitosis during treatment with CNDAC alone. These findings provide a mechanistic rationale for the design of new strategies, combining CNDAC with inhibitors of cell cycle checkpoint regulation in the therapy of hematologic malignancies.     

Cytotoxicity of N4-behenoyl-1-beta-D-arabinofuranosylcytosine through gradual conversion to 1-beta-D-arabinofuranosylcytosine in HeLa cells

The metabolism of 1-beta-D-arabinofuranosylcytosine (ara-C) and N4-behenoyl-1-beta-D-arabinofuranosylcytosine (BH-AC) was studied in Hela cells. After the cells were exposed to ara-C at a concentration of 20 micrograms/ml or BH-AC at 46.5 micrograms/ml for 1, 3, 6, 12 or 24 hr, the level of ara-C was determined using the radioimmunoassay method, and the level of BH-AC and 1-beta-D-arabinofuranosyluracil (ara-U), using high-performance liquid chromatography. In the ara-C-treated cells, the intracellular ara-C increased to 1.26 micrograms/g cells after exposure for 6 hr, and ara-C was rapidly changed to ara-U in the cells and in the medium. In the BH-AC-treated cells, the intracellular BH-AC increased after exposure for 24 hr and BH-AC was gradually converted to ara-C in the cells: the intracellular level of ara-C was only 15% of that of BH-AC after exposure for 24 hr. BH-AC level in the medium persisted for 24 hr, at the initial concentration. Our findings show that BH-AC is stable compared to ara-C and gradually converts to ara-C. This conversion is presumably a critical step in the antineoplastic effect of BH-AC.     

Targeting and anti-tumor efficacy of liposomal 5'-O-dipalmitoylphosphatidyl 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine in mice lung bearing B16BL6 melanoma

2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) is a potent anti-cancer agent, and we previously observed that liposomal formulation of 5'-O-dipalmitoylphosphatidyl derivative of CNDAC (DPP-CNDAC) is desirable for targeting. For targeting to pulmonary cancer, we investigated the in vivo behavior of liposomes containing DPP-CNDAC by a non-invasive method using positron emission tomography. Liposomes composed of DPP-CNDAC and cholesterol (DPP-CNDAC/CH liposomes) were markedly accumulated in mice lung bearing B16BL6 melanoma. In metastatic pulmonary cancer model, DPP-CNDAC/CH liposomes significantly reduced the lung colonization in a dose-dependent manner. The activity was significantly superior to conventional liposomal formulation or soluble CNDAC. These results suggest that DPP-CNDAC/CH liposomes are useful for metastatic pulmonary cancer.     

RACK1 regulates Ki-Ras-mediated signaling and morphological transformation of NIH 3T3 cells

Activating Ras mutations are involved in a significant fraction of human tumors. A suppressor screen using a retroviral mouse fibroblast cDNA library was performed to identify novel factors in Ras-mediated transformation. We identified a novel potent inhibitor of Ras-mediated morphological transformation encoded by a truncated version of the receptor for activated C-kinase (RACK1). The truncated protein, designated RACK1DeltaWD1, lacked the N-terminal 49 amino acids encoding the first of the 7 WD40 repeats in RACK1. RACK1DeltaWD1 expression restored contact inhibition, stress fiber formation and reduced ERK phosphorylation in Ki-Ras transformed NIH 3T3 cells. We demonstrate that truncated RACK1 is involved in complexes consisting of wild-type RACK1 and protein kinase C isoforms alpha, betaI and delta, compromising the transduction of an activated Ras signal to the Raf-MEK-ERK pathway. The cellular localization of RACK1DeltaWD1 differed from wtRACK1, indicating that signaling complexes containing the truncated version of RACK1 are incorrectly localized. Notably, 12-O-tetradecanoyl-13-phorbol acetate (TPA) mediated intracellular translocation of RACK1-interacting PKC alpha and delta was abrogated in RACK1DeltaWD1-expressing cells. Our data support a model where RACK1 acts as a key factor in Ki-Ras-mediated morphological transformation.     

Overexpression of Phosphatidylinositol Synthase Enhances Growth and G1 Progression in NIH3T3 cells

Phosphatidylinositol (PI) turnover is thought to play an important role in the regulation of cell growth. PI synthase (PIS, cytidine diphosphate (CDP)-diacylglycerol (DG): myo -inositol 3–phos–phatidyltransferase, EC 2.7.8.11) acts at the last step in the de novo biosynthesis of PI by catalyzing the condensation of CDP-DG and myo -inositol. To study the physiological role of PIS, we established murine NIH3T3 fibroblasts that stably overexpress PIS, by transfection with PIS cDNA (NIH-PIS cells). In immunofluorescence assays, the constitutively overexpressed PIS was found to be localized in the endoplasmic reticulum, as previously reported for the native enzyme activity. NIH-PIS cells showed an increase in PI synthesis in vitro and in vivo , as well as increased cellular levels of PI–4,5–P₂ and PI–3,4,5–P₃. They also displayed a decrease in their doubling tune and accelerated G1 progression. Overexpression of PIS increased cellular levels of the cyclin D1 and E proteins and Akt kinase activity in serum-stimulated quiescent NIH3T3 cells. Moreover, PIS Overexpression potentiated the colony formation of NIH3T3 cells in soft agar. These results suggest that PIS accelerates G1 progression and stimulates growth by increasing cellular levels of cyclins D1 and E.     

In vitro transformation of C3H/10T1/2 and NIH/3T3 cells by acrylonitrile and acrylamide

Acrylonitrile (AN) and acrylamide (AM) are carcinogenic in a number of rodent organs and AN is a suspected human carcinogen. We sought to determine whether AN and/or AM could produce morphological transformation in vitro in C3H/10T1/2 and NIH/3T3 mouse fibroblast cells. Both AN and AM induced a dose-dependent cytotoxic effect in C3H/10T1/2 and NIH/3T3 cells and readily transformed both cell lines. Our conclusions are based on the appearance of cells exhibiting a transformed phenotype and growth in soft agar. AN and AM transformed NIH/3T3 cells to a greater extent than C3H/10T1/2 cells. This is the first reported transformation of cells in vitro by AM.     

Antipain-induced suppression of oncogene expression in H-ras-transformed NIH3T3 cells

Antipain (AP; 50 micrograms/ml) inhibits transformation of NIH3T3 cells after transfection with an activated H-ras oncogene. To determine whether AP effects on transformation are associated with alterations in oncogene expression, NIH3T3 cells were cotransfected with an activated H-ras oncogene and the selectable marker gene aph, and gene expression was quantified. Fifty percent of geneticin-resistant colonies which were exposed to AP failed to express the transformed phenotype as determined by their inability to grow in soft agar. Northern blot analysis of the transformed and nontransformed colonies revealed that suppression of H-ras transformation by AP was associated with a decrease in expression of the exogenously transfected H-ras gene by approximately 4-fold. Expression of the endogenous oncogene c-myc was decreased by approximately 2.5-fold, to levels seen in untransfected cells. AP-treated colonies that retained the transformed phenotype had levels of oncogene expression that were similar to untreated ras-transformed colonies. Southern blot analysis revealed no effects of AP on incorporation or copy number of the H-ras gene.     

Hypersensitivity of NIH3T3 cells transformed by H-RAS gene to DNA-topoisomerase-I inhibitors

We examined the effects of the introduction of H-ras oncogene into murine cell line NIH3T3 on growth inhibition by topoisomerase-I (topo-I) inhibitors. The H-ras-transformed cells (pT22-3) showed approximately 12-fold increased sensitivity to a novel topo-I inhibitor, NB-506 [6-N-formylamino-12, 13-dihydro-I, II-dihydroxy-13-(β-D -glucopyranosyl)-5H-indolo(2,3-a)pyrrolo(3,4-c) carbazole-5,7(6H)-dione], compared with the parental NIH3T3 cells. pT22-3 also showed increased sensitivity to other topo-I inhibitors such as camptothecin (approx. 3.0-fold) and CPT-II (irinotecan, approx. 3.0-fold). Transformation of NIH3T3 by another oncogene (erb82) did not affect their sensitivity to these topo-I inhibitors. pT22-3 had approximately 32-fold higher topo-I activity than NIH3T3, but the same topo-I content. In a cell-free system, topo-I activity was increased 2-fold by addition of the H-ras protein precipitated from pT22-3 cells. Topo I in the nuclear extract of pT22-3 was eluted easily by low concentrations of NaCl compared with that of NIH3T3, suggesting a qualitative change in pT22-3 topo I. Increased phosphorylation of topo I was observed in pT22-3. Furthermore, NB-506 decreased the amount of the GTP-bound form of the H-ras product in pT22-3 cells. These results suggest that the high growth-inhibitory effect of a topo-I inhibitor, NB-506, on H-ras-transformed NIH3T3 cells is due to the H-ras-mediated signal-transduction pathway. © 1996 Wiley-Liss, Inc.     

Transformation of NIH3T3 cells with synthetic c-Ha-ras genes

Synthetic human c-Ha-ras genes in which amino acid codons were altered to those which are frequently used in highly expressed Escherichia coli genes were ligated to the 3'-end of Rous sarcoma virus long terminal repeat. When NIH3T3 cells were transfected with the plasmids having those genes with valine at codon 12, leucine at codon 61 or arginine at codon 61, transformants were efficiently produced. These results indicated that the synthetic c-Ha-ras genes are expressed in a mammalian system even though their codon usage is altered to correspond with that of E. coli. This expression vector system should be useful for studies on the structure-function relationships of c-Ha-ras, since the synthetic gene can be easily modified to have multiple base alterations, and can also be used simultaneously for the production of large amounts of p21 in E. coli for biochemical and biophysical studies.     

Effect of glucocorticoids on oncogene transformed NIH3T3 cells

We studied the differential response of oncogene transformed NIH3T3 cells to glucocorticoids. As demonstrated for transformed human fibroblasts, the morphology of neu-, ras-, src- and sis-transformed mouse fibroblasts became more normal after glucocorticoid treatment. This change was not due to inhibition of the expression of oncogene mRNA or protein. However, the abl-transformed NIH3T3 cells were resistant to glucocorticoid-induced morphology change. These results indicate that the glucocorticoid-induced morphology change is specific to certain oncogene-transformed NIH3T3 cells. Transformed human fibroblasts generally have reduced amounts of cell surface fibronectin. When treated with glucocorticoids, they incorporate higher levels of fibronectin in their extracellular matrix, which correlates with their change in morphology. However, we found that, except for abl-transformed cells, the fibronectin level of the other oncogene transformed mouse cells was similar to non-transformed cells. Moreover, treatment of the neu-, ras-, src- and sis-transformed cells with glucocorticoids resulted in a change in morphology but no increase in cell surface fibronectin. These studies demonstrate that the glucocorticoid-induced morphological change of oncogene-transformed NIH3T3 cells is not due to enhanced expression of fibronectin. Therefore, other mechanisms are responsible for this glucocorticoid-induced phenotypic change of oncogene-transformed cells.     

Midkine induces the transformation of NIH3T3 cells.

Midkine (MK) is a heparin-binding growth factor and is frequently expressed at high levels in many human carcinomas. To investigate further the roles of MK in the regulation of cell growth, we introduced MK expression in NIH3T3 cells. A mixture of transfectants of an MK expression vector, but not a control vector, formed colonies in soft agar, showed an elevated cell number at confluence, and formed tumours in nude mice. An interesting characteristic of the transformed cells was that they became spontaneously detached from the culture dish substratum. In the transformed cells, MK was not only secreted, but also localized, in the perinuclear region as spots. The present data indicate that MK has the potential to transform NIH3T3 cells and suggest that overexpression of the MK gene may promote unregulated cell growth in vivo.     

Overexpression of phosphatidylinositol synthase enhances growth and G1 progression in NIH3T3 cells.

Phosphatidylinositol (PI) turnover is thought to play an important role in the regulation of cell growth. PI synthase (PIS, cytidine diphosphate (CDP)-diacylglycerol (DG): myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) acts at the last step in the de novo biosynthesis of PI by catalyzing the condensation of CDP-DG and myo-inositol. To study the physiological role of PIS, we established murine NIH3T3 fibroblasts that stably overexpress PIS, by transfection with PIS cDNA (NIH-PIS cells). In immunofluorescence assays, the constitutively overexpressed PIS was found to be localized in the endoplasmic reticulum, as previously reported for the native enzyme activity. NIH-PIS cells showed an increase in PI synthesis in vitro and in vivo, as well as increased cellular levels of PI-4,5-P2 and PI-3,4,5-P3. They also displayed a decrease in their doubling time and accelerated G1 progression. Overexpression of PIS increased cellular levels of the cyclin D1 and E proteins and Akt kinase activity in serum-stimulated quiescent NIH3T3 cells. Moreover, PIS overexpression potentiated the colony formation of NIH3T3 cells in soft agar. These results suggest that PIS accelerates G1 progression and stimulates growth by increasing cellular levels of cyclins D1 and E.     

Transformation of NIH3T3 cells by transfection with UV-irradiated human c-Ha-ras-1 proto-oncogene DNA

Our previous studies have shown that human skin cancers occurring on sun-exposed body sites frequently contain G----T mutations at the second position of Ha-ras codon 12. In this study, we investigated whether the c-Ha-ras-1 proto-oncogene could be activated by in vitro UV-irradiation of pEC plasmid DNA, which contains the 6.6 kb BamHI fragment of the human c-Ha-ras-1 proto-oncogene. Focus formation and nude mouse tumorigenicity assays showed that UV-irradiated pEC DNA induced morphologic and tumorigenic transformation of NIH3T3 cells in multiple cycles of transfection, whereas unirradiated pEC DNA did not. DNAs from secondary cycle foci and tertiary cycle tumors were analyzed for mutations in Ha-ras codons 12 and 61 using the polymerase chain reaction and synthetic oligonucleotide probes. Eleven of 11 secondary cycle foci analyzed possessed a G----T mutation at the second position of Ha-ras codon 12. However, the nude mouse tumors exhibited a G----A mutation at position 1 of the Ha-ras codon 12. These results suggest that in vitro UV irradiation of the c-Ha-ras-1 proto-oncogene DNA can induce mutations that are similar to those found in human skin cancers that originated on sun-exposed body sites.     

Saturation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate accumulation in leukemia cells during high-dose 1-beta-D-arabinofuranosylcytosine therapy

Twenty-seven patients with refractory leukemia were treated with 1-beta-D-arabinofuranosylcytosine (ara-C), 0.3 to 3.0 g/m2 as i.v. infusions over 1, 2, 4, or 24 h. The pharmacokinetics of ara-C in plasma and its 5'-triphosphate (ara-CTP) in leukemic cells from peripheral blood were studied after a single infusion of 3 g/m2 over 2 h in 13 patients. Accumulation of ara-CTP in leukemic cells remained linear until 1 to 2 h after the infusion. At the time when the rate of ara-CTP accumulation deviated from linearity, the plasma concentration of ara-C was 5- to 20-fold lower [8.1 +/- 4.4 (SD) microM] than the steady-state level during the infusion. Plasma ara-C and cellular ara-CTP pharmacokinetics were studied after two serial infusions in 14 additional patients. Varying the duration of infusion of an ara-C dose between 1, 2, and 4 h (corresponding to infusion rates of 3000, 1500, and 750 mg/m2/h) did not substantially change the rate of ara-CTP accumulation by leukemic cells. The peak ara-CTP concentration and the area under the concentration times time curve (AUC) of ara-CTP in leukemic cells increased with prolongation of the infusion. Although steady-state concentration of ara-C and AUC of ara-C in plasma were proportionally reduced by 1.0 or 0.5 g/m2 infusion over 2 h, ara-CTP accumulation rate and AUC in leukemic cells did not change compared with administration of 3 g/m2 over 2 h. However, when the infusion rate was further reduced to 0.4 or 0.3 g/m2 over 2 h, resulting in steady-state plasma ara-C concentrations of less than 7 microM, the accumulation rate of ara-CTP was substantially reduced as was the ara-CTP intracellular AUC. The cellular elimination rate of ara-CTP remained constant under all infusion conditions. These findings support the conclusion that high-dose ara-C therapy, as currently administered, results in plasma ara-C concentrations that saturate the accumulation of ara-CTP by circulating leukemic cells. We recommend that intermediate dose rates, 200 to 250 mg/m2/h, be evaluated in future studies as an alternative to the substantially higher ara-C dose rates currently in use.