Cytotoxic effects of a novel maleimide derivative on epithelial and tumor cells

Novel N-triazolyl maleimide derivatives were synthesized by azide–alkyne Huisgen cycloaddition (1,3-dipolar cycloaddition) and tested for cytotoxicity against a cell line derived from human melanomas SK-Mel-28 and SK-Mel-103, and human umbilical vein endothelial cell lines (HUVEC). The 4l was chose to be biologically tested due to incorporation of benzyl triazolic to the nitrogen of maleimide has not been tested before, and due the satisfactory yield. The analysis of cell metabolism, using the MTT method, showed that the compound 4l impaired cell metabolism in HUVEC only in high concentration (100 µM). A lower concentration of compound 4l, whether in association or not with paclitaxel, was required to cause toxicity in both SK-Mel-28 and SK-Mel-103 cells in comparison with HUVEC cells. Moreover, the ability of 4l to cause cell death was evaluated by flow cytometry, and the data obtained highlighted the apoptotic action of 4l and paclitaxel co-treatment on Sk-Mel-28 cells only, which corroborated the greater efficacy of maleimide compounds against cancer cells. Together, our data provide promising data on the selectivity of maleimide compounds to cancer cells, and suggest that novel maleimide-substituted compounds may be synthesized and tested on different cancer cell lines, as primary or co-adjuvant agents of cancer cell toxicity.     

Influence of the deprivation of a single amino acid on cellular proliferation and survival in rat 3Y1 fibroblasts and their derivatives transformed by a wide variety of agents

We compared proliferation and survival of various syngeneic transformed cell lines under conditions of depletion of 15 amino acids in Dulbecco-Eagle's medium. We used a normal fibroblast line 3Y1 and 22 transformed sublines of 3Y1 which had been induced by one of seven transforming agents--simian virus 40, mouse polyomavirus, adenovirus type 12, E1A gene of adenovirus type 12, cDNA of Harvey murine sarcoma virus, Rous sarcoma virus, or N-methyl-N'-nitro-N-nitrosoguanidine. Unlike other untransformed cells examined (mouse BALB/c-3T3 line, mouse NIH-3T3 line, and primary Fischer rat embryo fibroblasts), 3Y1 ceased to proliferate and accumulated in a viable state with a G1-phase DNA content under 14 singular deprivations of amino acid. None of the transformed 3Y1 lines completely arrested in the G1 phase of the cell cycle and each showed different levels of survival, depending on each transforming agent. As for transformed 3Y1 cells induced by a given virus or a given transforming gene, any one of the three sublines shared the same trend with respect to proliferation and survival. Transformed derivatives induced by N-methyl-N'-nitro-N-nitrosoguanidine showed almost the same trend in proliferation, but the patterns of survival were not uniform. Our observations suggest that the unique responses of 3Y1 to amino acid depletion are differently modified by different transforming agents.     

N-(1-Pyrenyl) maleimide inhibits telomerase activity in a cell free system and induces apoptosis in Jurkat cells

Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. Agents that interact selectively with telomerase are anticipated to exert specific action on cancer cells. In this study, we evaluated maleimide derivatives for their potency and selectivity of telomerase inhibition. Among the several N-substituted derivatives of maleimide tested, N-(1-Pyrenyl) maleimide was shown to exert the greatest inhibition of telomerase in a cell free system, with an IC50 value of 0.25 μM. Importantly, we demonstrated that N-(1-pyrenyl) maleimide induces apoptosis in Jurkat T cells and displays the greatest differential cytotoxicity against hematopoietic cancer cells. These results suggest that N-(1-pyrenyl) maleimide is an attractive maleimide to be tested and developed as anti-cancer drug.     

Consistent oncogene methylation changes in epithelial cells chemically transformed in vitro

We have examined the restriction digest patterns of CCGG sequences in Kiras, Ha-ras, and c-myc oncogenes in rat tracheal epithelial cells transformed in vitro by 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene/12-O-tetradecanoylphorbol-13-acetate (TPA), or TPA alone. Oncogenes c-myc and Ha-ras in transformed cell lines, compared to normal rat tracheal epithelial cells and untreated primary cultures, had altered Hpa II restriction patterns as demonstrated by hybridizing bands of different molecular weight, or loss of bands. Ki-ras was hypermethylated in all cell derivations, including normal cells. These molecular alterations have not previously been reported for epithelial cells transformed in vitro by polycyclic hydrocarbons and tumor promoters, and suggest common mechanisms of action for agents with diverse molecular targets.     

Production of plasminogen activator by established cell lines of mouse origin

The correlation between malignant transformation and increased plasminogen activator synthesis has been studied in a variety of established cell lines. In contrast to the behavior of secondary mouse embryo cultures, which always show increased fibrinolytic activity when transformed, no such correlation was found within the BALB/c 3T3 line and its transformed derivatives. Cell lines were established from tumors initiated in BALB/c mice by several transformed cell lines. These lines were generally found to contain no more plasminogen activator than the cells used for inoculation. A correlation was found between transformation and plasminogen activator synthesis within Swiss 3T3 cell lines. However, the correlation was not maintained by serum revertants of transformed Swiss 3T3 cells.     

Altered ganglioside composition in virally transformed rat embryo fibroblasts.

The composition of gangliosides was examined in a normal rat embryo fibroblast cell line (REF52) and in two viral transformants: a polyoma transformant (REF52-PyMLV) and a simian viral 40 transformant (REF52-SV40). The distribution of gangliosides in the cell lines was determined using gas-liquid chromatography and high-performance thin-layer chromatography. N-acetylneuraminic acid was the predominant sialic acid species detected in the three cell lines. The total ganglioside concentration (microgram/100 mg dry weight of cells) in the normal, PyMLV, and SV40 lines was 144.7 +/- 10.4, 153.8 +/- 9.2, and 86.1 +/- 6.8, respectively. Gangliosides GM3, GM2, GM1, and GD1a were the major species in the normal and transformed lines. The distribution of these gangliosides, however, differed markedly between the normal and the transformed lines and also between the transformed lines themselves. The transformed cells also differed from the normal cells in growth rate, morphology, and social behavior. The cell line with highest GM3 content (PyMLV) formed islands, whereas the normal and SV40 cell lines, which had lower GM3 levels, grew as monolayers. The findings suggest that PyMLV and SV40 transformation can have multiple and different effects on cellular ganglioside distribution and growth behavior.     

Insulin inhibition of protein degradation in cell monolayers

Protein degradation has been measured in confluent monolayers of eleven lines of contact-inhibited cells and ten transformed lines as the rate of release of trichloroacetic acid-soluble radioactivity after prelabeling cell protein with [³H]leucine. Insulin, at concentrations from 10^?12 M to 10^?6 M, has been added at the beginning of the 4-hour degradation period to detect selective effects of this hormone as an inhibitor of the inducible proteolysis occurring in serumfree medium. In addition insulin binding measurements have been performed on selected cell lines in an attempt to relate receptor properties to insulin action. Substantial effects of insulin are found in most cells with a selective inhibition at low insulin concentrations noted in several of the transformed lines. The difference in insulin sensitivity is not entirely definitive because temperature-sensitive transformation mutants of NRK cells are not more sensitive to insulin at a temperature where they show the transformed phenotype. Although insulin receptors on different cell lines have similar binding properties, two of the hepatomas used, H35 and MH₁C₁, show inhibition of protein degradation at insulin concentrations where receptor occupancy is extremely low. Calvarial osteoblast-like cells have a high rate of protein degradation which can be reduced by growth factors but not by insulin. The lack of an insulin response is a consequence of poor insulin binding to the cells. Insulin binds to the osteogenic sarcoma cells in substantial amounts. However, its normal action to inhibit the induced proteolysis is restricted because with these cells no increase of proteolysis occurs in serum-free medium. Generally higher rates of protein degradation are observed in the contact-inhibited lines than the transformed cells. We suggest that this difference may provide a selective growth advantage to transformed cells.     

DNA methylation in mouse cells in culture as measured by restriction enzymes

Methylation of DNA in normal mouse cultured 3T3 cells and in their virally or chemically transformed derivatives was studied. DNA methylation was studied by restriction with HpaII, MspI, or HpaII plus MspI. DNA from the chemically transformed cells was cleaved about twice as often with HpaII than was the DNA of normal and virally transformed cells. Digests with MspI and HpaII plus MspI were identical in all cell lines studied. Densitometry of the restriction patterns allowed an estimate of total DNA methylation from the weight average lengths. The chemically transformed cell line showed 25% reduction in methylation compared to the other cell lines. Southern blot hybridization using satellite DNA showed that these sequences followed a pattern of modification similar to that of total DNA.     

Transformed cells have lost control of ribosome number through their growth cycle.

Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus-transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in a post-mitotic pre-DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability. The rate of protein synthesis in the stationary phase of the transformed cells fell to 25-30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribisome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L-73 and LR-73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells. A model for growth control of normal cells and its derangement in transformed cells is presented.     

A G2/M cell cycle block in transformed cells by contact with normal neighbors

Neighbour suppression of growth of tumour cells by stationary normal cells might be important in early stages of cancer. We have studied this using suppressor and non-suppressor lines of 3T3 fibroblasts and SV40 transformed derivatives. Growth suppression of transformed cells depended on direct contact with stationary confluent cultures of 3T3 cells but not on gap junction communication. It was not caused by apoptosis nor through the normal G0/G1 block present in the confluent normal cells. Instead, there was a progressive elongation of the cell cycle leading to arrest in G2/M in the transformed cells. This indicates an unusual type of growth arrest not previously involved in social control of cell growth.     

Aggregation of normal and transformed cells attached to a substrate]

On the surface of the nirtocellulose membrane filter (pore size 0.3--0.5 mem), normal mouse or hamster embryo fibroblasts formed discrete cell aggregates. Behaviour of transformed fibroblast-like cells of 9 different lines was compared with that of normal cells. Cells of 3 transformed lines grew on this substratum as a uniform monolayer displaying no tendency to aggregation. The following 3 cell lines exposed a slightly "patchy" cell distribution on the 3rd--4th day of cultivation but were unable to form discrete aggregates. The remaining 3 lines did form aggregates but the dynamics of aggregation and the final aggregation pattern for two of them were abnormal. Only one of the 9 investigated transformed lines had the normal aggregation behaviour. Hence, in the course of the neoplastic evolution, cells lose their ability fo form aggregates on the filter surface. Mechanisms of cell aggregation and possible reasons of differencies in the aggregation behaviour between normal and transformed cells, are discussed.     

Behavior of cells seeded in isolated fibronectin matrices

Cell-free fibronectin matrix (FN-matrix) isolated from chick embryo fibroblasts was used to study cell-matrix interaction. After 24 h, most fibroblastic cells, including those without cell surface fibronectin, adopted bipolar fusiform morphology. Cells grew in parallel arrays and aligned with each other apparently along FN-matrix. Since the orientation of fibronectin fibers was determined by chick embryo fibroblasts, our results suggested that intercellular organization of "matrix-using" cell type may be influenced by "matrix-producing" cell type. Whereas the elongation and alignment effects induced by FN-matrix have been detected in fibroblasts (both normal and transformed), myoblast, aortic endothelial cells, neural cell lines (B103 and RT4D1), and cardiac muscle cells, similar effects are not detected in bone marrow hemopoietic cells, circulating lymphocytic T and B cells, and sympathetic neurons. For epithelial cells, FN-matrix has varying effects. Elongation and alignment effects are detected only in transformed epithelial cells with a great reduction in keratin expression. The morphology of normal or transformed epithelial cells with abundant keratin appears unaffected by FN-matrix. FN-matrix reduced the growth of several transformed fibroblastic lines up to 25%, but did not restore the appearance of actin stress fibers and the normal migratory activities of Rous sarcoma virus-transformed rat cells.     

Synthesis and biological evaluation of novel benzodioxinocarbazoles (BDCZs) as potential anticancer agents

We report the efficient synthesis and biological evaluation of new benzodioxinoindolocarbazoles heterocycles (BDCZs) designed as potential anticancer agents. Indolic substitution and maleimide variations were performed to design a new library of BDCZs and their cytotoxicity were evaluated on two representative cancer cell lines. Several derivatives have shown a marked cytotoxicity with IC₅₀ values in the nanomolar range. Results are reported in this Letter.     

Differential responses of lignin derivatives between tumor and normal tissue derived cell lines; effects on cellular adhesion and cell growth.

When cell lines derived from tumor tissues were plated simultaneously with the lignin derivative or dextran sulfate in plastic culture dishes, the cells did not completely adhere to dishes and/or the shape of even the adhering cells changed significantly. On the other hand, using cell lines derived from normal tissues, no significant effect of the polyanions was observed. The present study revealed that the lignin derivatives demonstrated differential responses between cells derived from tumor tissues and from normal tissues, and that the lignin derivatives strongly inhibited the growth of mouse sarcoma cells (FRUKTO) and rat foetal cells (Ad12-3Y1-Z19) transformed with adeno virus type 12.     

Cellular compartmentation of calcium in mouse fibroblasts in vitro depends on cell density and transformation

Cellular compartmentation of Ca has been investigated by kinetic analysis of 45Ca efflux from preloaded cells at various states of cell density-dependent proliferation of normal (3T3) and transformed (3T6 and SV40-3T3) mouse cells. Three pools of exchangeable calcium were separated on the basis of their differing exchange kinetics. For each of the cell lines tested, all three compartments decrease with cell density. Significant differences between normal and transformed cells are observed upon quiescence of the normal cells, where the slowly exchanging compartment of normal cells gradually increases, whereas that of the transformed cells continues to decrease (with increasing cell density). Free cytoplasmic Ca2+ concentration as determined by the Quin 2 method, was found to be significantly higher in transformed cells than in normal cells. These results indicate significant differences in Ca homeostasis between normal and transformed cells.     

Transformation by the k-ras oncogene correlates with increases in phospholipase A2 activity, glycerophosphoinositol production and phosphoinositide synthesis in thyroid cells.

Two cell lines transformed by the k-ras oncogene (KiKi and KiMol cells) and a temperature sensitive clone (Ts), all originated from a normal rat thyroid line (FRTL5 cells), have been employed to analyse the intracellular mechanisms affected by the ras p21. In k-ras transformed cells two phosphoinositide derivatives, glycerophosphoinositol and inositol monophosphate, were markedly increased, whereas inositol bisphosphate and trisphosphate maintained the same level as in normal cells. Cytosolic Ca2+ was also unaffected. This indicates that in epithelial cells the phospholipase C activity is not altered upon ras transformation. The formation of glycerophosphoinositol involved the activation of a phosphoinositide specific phospholipase A2. The higher phospholipase A2 activity in ras transformed cells could be further demonstrated by the increase in total arachidonic acid release. In the Ts clone the increase in glycerophosphoinositol and inositol monophosphate was evident only at the permissive temperature (33 degrees C), whereas it disappeared at 39 degrees C. At 33 degrees C the cells were also characterized by an enriched membrane pool of phosphoinositides. All these changes occurred in parallel with morphological transformation. We propose that cell transformation by the k-ras oncogene affects different steps of the membrane lipid metabolism, among which the most prominent one is the activation of a phosphoinositide specific phospholipase A2. These effects could originate mitogenic metabolites. Moreover, they correlate well with the induction of the malignant phenotype.     

Identification in several human myeloid leukemias or cell lines of a DNA rearrangement next to the c-mos 3'-end

A characteristic DNA rearrangement, the loss of an EcoRI cleavage site next to the 3'-end of the human c-mos gene, has been found to be frequently present in DNA from transformed hematopoietic cells of the myeloid lineage but not in DNA from either normal or transformed cells of different tissue types. Three established cell lines, respectively a pro-monocytic line (CM-S) and two precursor granulocytic lines (My/K1 and My/K5), carry the same genome rearrangement, but not fibroblasts obtained from the marrow of the same patients. This DNA rearrangement is maintained in three different hybridomas derived by fusion of CM-S cells with normal human embryo hepatocytes.     

Formation and utilization of methionine by rat liver cells in culture

The enzyme N5-methyltetrahydrofolate:homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells.     

Steroids and the prostate

Interelationships between steroid and growth factor regulation of cell proliferation has been examined in two androgen sensitive prostatic cell lines, grown in defined medium. The cell lines used were derived from normal (CAPE) and neoplastic (LNCaP) tissues. The growth of both cell lines was elevated by challenge with serum, androgens and epidermal growth factor (EGF) used as single agents. The effects of androgen in CAPE were small, but significant while the profound effects of these agents on the growth of LNCaP were confirmatory of other studies. Androgens upregulated EGF receptor expression in LNCaP measured by both ligand binding capacity and mRNA analysis. This was not observed in the CAPE cells. Addition of serum (whole or charcoal stripped) suppressed the observed androgenic stimulation of EGF receptor expression in LNCaP. This apparent anomaly is discussed in relation to the growth enhancing properties of serum in these cell lines and in the wider context of normal and neoplastic growth control in the prostate.