Formation Of Transformed Foci Research Articles

Deprivation of glutamine in cell culture reveals its potential for treating cancer

The growth-stimulating capacity of calf serum (CS) in cell culture reaches a maximum of 10% with Balb 3T3 cells, remains at a plateau to 40% CS, and declines steeply to 100% CS. Growth capacity can be largely restored to the latter by a combination of cystine and glutamine. Glutamine is a conditionally essential amino acid that continues to function at very low concentrations to support the growth of nontransformed cells, but transformed cells require much larger concentrations to survive. These different requirements hold true over a 10-fold variation in background concentrations of CS and amino acids. The high requirement of glutamine for transformed cells applies to the development of neoplastically transformed foci. These observations have given rise to a novel protocol for cancer therapy based on the large difference in the need for glutamine between nontransformed and transformed cells. This protocol would stop the cumulative growth and survival of the transformed cells without reducing the growth rate of the nontransformed cells. The results call for studies of glutamine deprivation as a treatment for experimental cancer in rodents and clinical trials in humans.

NIPA Checkpoint Control In Oncogenic Transformation Is Dependent on p53

AbstractAbstract 4178The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. Using a conditional knockout strategy we inactivated the gene encoding NIPA. NIPA-deficient animals are viable, but sterile due to a block of spermatogenesis. Moreover, our studies demonstrate that loss of NIPA has no substantive effect on the physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, NIPA checkpoint control can be unmasked by oncogenic transformation by c-Myc. Here we show that transformed focus formation assays revealed highly significant differences in c-Myc-induced transformation in NIPA-deficient and wild-type MEFs. c-Myc transduction caused a pronounced upregulation of cyclin-B in NIPA-null MEFs, which was completely reversible by ectopic NIPA expression. The increased cyclin-B1 expression after c-Myc transduction in the absence of NIPA has considerable functional consequences for the cells: Focus formation ability of c-Myc-infected Nipa-/- MEFs was greatly reduced in comparison to wild-type MEFs (24.6% vs. 100%). Moreover, c-Myc expression caused 12.8% apoptotic subG1 cells in wild-type MEFs, whereas Nipa-/- MEFs were more affected by c-Myc-induced apoptosis (22.45%). Next, we sought to know, whether increased apoptosis in Nipa-deficient c-Myc transduced MEFs is dependent on a functional p53-Axis. Therefore, Nipa-wildtype and knockout MEFs were first infected with a retroviral Supernatant encoding for a p53Mir- and thereafter with the oncogene c-Myc. Interestingly the effect of Nipa knockout on c-Myc-mediated oncogenic transformation was totally abolished by the knock-down of p53. We observed no differences in focus formation ability or growth behaviour in Nipa-/- MEFs with inactivated p53 in comparison to wildtype cells, suggesting the importance of functional p53 in Nipa-induced cell death. Taken together, our data demonstrate that NIPA is required for efficient c-Myc transformation in a p53-dependent manner. Moreover, our results highlight the functional importance of the NIPA-p53 axis in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis. Disclosures:No relevant conflicts of interest to declare.

Analogues of Y27632 increase gap junction communication and suppress the formation of transformed NIH3T3 colonies

Background:Constitutive activation of RhoA-dependent RhoA kinase (ROCK) signalling is known to promote cellular transformation and the ROCK inhibitor Y-27632 has the ability to suppress focus formation of RhoA transformed NIH3T3 cells.Methods:Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras. In vitro kinase inhibitor profiling, co-culture of transformed cells with non-transformed cells and a novel Lucifer yellow/PKH67 dye transfer method were used to investigate their mode of action.Results:Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn. No toxicity was observed against either transformed or non-transformed cells and the effect was dependent on co-culture of these two cell types. In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4). Dye transfer analysis showed they increased gap junction intercellular communication (GJIC) between transformed and non-transformed cells.Conclusions:These data are the first to suggest that transient blockade of specific kinases can induce a persistent inhibition of non-contact inhibited transformed colony formation and can also remove pre-formed colonies. These effects could potentially be mediated by the observed increase in GJIC between transformed and non-transformed cells. Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.

NIPA Checkpoint Control Is Essential for Efficient Oncogenic Transformation by C-Myc

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. Using a conditional knockout strategy we inactivated the gene encoding NIPA. NIPA-deficient animals are viable, but sterile due to a block of spermatogenesis. Moreover, our studies demonstrate that loss of NIPA has no substantive effect on the physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, NIPA checkpoint control can be unmasked by oncogenic transformation by c-Myc. Here we show that transformed focus formation assays revealed higly significant differences in c-Myc-induced transformation in NIPA-null and wild-type MEFs. c-Myc transduction caused a pronounced upregulation of cyclin-B in NIPA-null MEFs, which was completely reversible by ectopic NIPA expression. This increased cyclin-B1 expression after c-Myc transduction in the absence of NIPA has considerable functional consequences for the cells: Focus formation ability of c-Myc-infected Nipa−/− MEFs was greatly reduced in comparison to wild-type MEFs (24.6% vs. 100%). Moreover, c-Myc expression caused 12.8% apoptotic subG1 cells in wild-type MEFs, whereas Nipa−/− MEFs were more affected by c-Myc-induced apoptosis (22.45%). By contrast, transduction with other oncogenes like k-Ras in p53 knockdown Nipa−/− and Nipa+/+ MEFs showed no differences in various transformation and apoptosis assays pointing out the exclusive role of the G2/M checkpoint NIPA in c-Myc induced transformation. Furthermore, we investigated the impact of these findings for the pathogenesis of c-Myc induced tumorigenesis in vivo. Recipient mice transplanted with c-Myc transduced wild-type bone marrow rapidly developed an AML-like disease (median survival 33 days) characterized by bone marrow infiltration and expression of the myeloid lineage markers CD11b and Gr1. In contrast, animals transplanted with c-myc transduced NIPA knockout BM showed a substantially delayed onset of leukemia and survived significantly longer compared to the control group (median survival 52 days, p<0.01). Taken together, our data demonstrate that NIPA is required for efficient c-Myc transformation in vitro and and in vivo in a murine bone marrow transplantation model. Moreover, our results highlight the functional importance of NIPA in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis.

Degrees and kinds of selection in spontaneous neoplastic transformation: an operational analysis.

Spontaneous neoplastic transformation develops within days in the NIH 3T3 line of cells through differential inhibition of their proliferation under contact inhibition. A small fraction of the population continues to multiply after saturation density is reached and is selected to progressively increase saturation density in successive rounds of confluence. The degree of selection at confluence depends on the extent of proliferation of some cells in a heterogeneous population. The development of transformed foci is an extension of the same selective process that increases saturation density. The expression of the foci is enhanced with increases in the saturation density of the surrounding cells. Transformation is also induced by moderately reducing the concentration of calf serum in the medium during low-density passages, which allows selection of cells that require less growth factor. Further stepwise reductions in serum increase the degree of transformation. Contact inhibition and reduction in serum concentration select for the same phenotype of cell that increases saturation density and generates transformed foci. There is mounting evidence that selection is a major factor in the development of common epithelial tumors of humans, but it extends over decades rather than days, and the in vivo microenvironment selects from more stable populations of cells than those in culture. The many progressive levels of increased saturation density and transformed focus formation suggest that a very large number of genes participate in neoplastic development. The operational model of variation and selection presented here may aid in understanding chemical carcinogenesis and cancer recurrence after chemotherapy.

Heritable, population-wide damage to cells as the driving force of neoplastic transformation.

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in the death of some cells in a manner suggestive of apoptosis. Successive rounds of prolonged incubation at confluence of the surviving cells produce increasing neoplastic transformation in the form of increments in saturation density and transformed focus formation. Cells from the postconfluent cultures are given a recovery period of various lengths to remove the direct inhibitory effect of confluence before their growth properties are studied. It is found that with each round of confluence the exponential growth rate of the cells at low densities gets lower and the size of isolated colonies of the same cells shows a similar progressive reduction. The decreased growth rate of cells from the third round of confluence persists for > 60 generations of growth at low density. The proportion of colonies containing giant cells is much higher after a 2-day recovery from confluence than after a 7-day recovery. Retardation of growth at low density and increased saturation density appear to be two sides of the same coin: both occur in the entire population of cells and precede the formation of transformed foci. We propose that the slowdown in growth and the formation of giant cells result from heritable damage to the cells, which in turn drives their transformation. Similar results have been reported for the survivors of x-irradiation and of treatment with chemical carcinogens and are associated with the aging process in animals. We suggest that these changes result from free radical damage to membrane lipids with particular damage to lysosomes. Proteases and nucleases would then be released to progressively modify the growth behavior and genetic stability of the cells toward autonomous proliferation.

Carbonic anhydrase is abundant in fenestrated capillaries of cherry hemangioma.

The ability of dithranol and 10-butyryl dithranol to induce morphological cell transformation was studied in cultured C3H 10T1/2 C18 fibroblasts. The cells were incubated with different concentrations of the test compounds for 48 h and cultured for 5 weeks thereafter. At the end of the culture period the cultures were fixed, stained and examined for the presence of transformed foci. Dithranol and 10-butyryl dithranol did not increase the formation of transformed foci, while the positive control compound, 7,12-dimethylbenz(a)anthracene (DMBA), induced a high frequency of transformations significantly different from controls. Thus the in vitro cell transformation model with uninitiated C3H 10T1/2 C18 fibroblasts is not able to detect the weak tumorigenic action of dithranol and 10-butyryl dithranol which has been observed in mouse skin.

The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins.

Cooperation between the adenovirus E1A and E1B oncogenes is required for transformation of primary quiescent rodent cells. Although expression of E1A alone will stimulate cell proliferation sufficient to initiate transformed focus formation, proliferation fails to be sustained and foci degenerate. Coexpression of either the 19-kDa or 55-kDa E1B oncoproteins with E1A permits high-frequency transformation by overcoming this cytotoxic response. Without E1B 19-kDa protein expression, however, transformants remain susceptible to induction of cell death. Rapid loss of viability is coincident with nucleolytic cleavage of DNA in intranucleosomal regions and chromatin condensation, hallmarks of programmed cell death (apoptosis). Furthermore, overexpression of a known suppressor of apoptosis, the Bcl-2 protooncogene, can rescue E1A-induced focus degeneration. Thus E1A-dependent stimulation of cell proliferation is accompanied by apoptosis and thereby insufficient to singly induce transformation. High-frequency transformation requires a second function encoded by the E1B 19-kDa protein to block apoptosis.

Effects of retinoic acid on NIH3T3 cell transformation by the H-ras oncogene

Exposure of NIH3T3 cells to retinoic acid resulted in a dose-dependent modulation of transformed focus formation after transfection with an activated H-ras oncogene. Inhibition induced by 10 microM retinoic acid was maximal at 21.4% of control values. Maximal inhibition of transformation was found after exposure to 10 microM retinoic acid between days 0 and 3 of the transfection period. This concentration was also inhibitory for colony formation upon transfection of the non-transforming gene aph, suggesting that retinoic acid acts primarily on the process of transfection to inhibit focus or colony formation. Exposure to retinoic acid during the late period of the transfection protocol (days 14-20) resulted in alterations in focus morphology. A transformed cell line containing H-ras underwent reversion of the transformed phenotype after 4 weeks of treatment with retinoic acid, as determined by alterations in cell morphology and anchorage-independent growth. Phenotypic reversion was not associated with changes in the expression of the exogenous H-ras or endogenous c-myc or c-fos oncogenes.