Synthesis and in vitro antitumor activity of asperphenamate derivatives as autophagy inducer

In an effort to improve the aqueous solubility and the antitumor activity of natural product asperphenamate, we have designed and synthesized three series of asperphenamate derivatives, including series I (simplifying molecular skeleton series), series II (introducing a hydroxyl group to A-phenyl ring series) and series III (disrupting molecular planarity series). All derivatives have displayed a significantly increased solubility compared with asperphenamate. Their growth inhibitory activities in vitro were screened by the standard MTT method in MCF-7, HeLa, and BEL-7402 cell lines. With the exception of the derivatives in series I, most of derivatives in series II and series III showed growth inhibitory activity. Among all derivatives, IM23b in series III showed the greatest potency in human breast cancer MCF-7 cells. The cellular potency of IM23b was approximately 1.5-fold more potent than that of cisplatin. The mechanism of cell death induced by IM23b in human breast cancer MCF-7 cells was further investigated. We concluded that the cell death was induced by autophagy instead of apoptosis or cell cycle arrest.     

Step-fortifications of nutrients in mammalian cell culture

A series of high-density media for mammalian cell culture were developed by step-fortifications of most nutrient components in RPMI-1640 medium. Each medium constituting the series was constructed to meet in vitro cell growth limitations. Four different cell lines were cultivated in the media series, and their growth characteristics were observed. Maximum cell densities varied in the range of 0.4 to 1.3 x 10(7) cells/mL, depending on cell lines. Cell growth responses to each of the media series were analyzed in terms of cell density and cell mass. Step increases of cell mass in the range of 1.3 to 3.7 g/L were observed according to the step-fortifications of nutrients. Also, the characteristics of each cell line were compared in terms of metabolic yields and specific productions of lactic acid and ammonium ion. The effect of step-fortifications of nutrients on the production of monoclonal antibody was also examined. Apparent differences in metabolic characteristics among cell lines were observed. Experimental results suggested that the different cell sizes and metabolic characteristics of each cell line resulted in cell-line-specific responses to the step-fortifications. The significant influence of nutritional fortifications on high-density culture of mammalian cells was evaluated. (c) 1993 John Wiley & Sons, Inc.     

Lack of correlation between the decreased expression of cell surface LETS protein and tumorigenicity in human cell hybrids

An analysis of the correlation between tumorigenicity and the loss of expression of the large external transformation-sensitive glycoprotein (LETS) was performed on human cell hybrids and their respective normal and tumorigenic parental cell lines. The distribution of cell surface LETS protein in a series of cell lines was examined by both specific immunofluorescent staining and by gel electrophoresis of lactoperoxidase-catalyzed, iodinated cell surface proteins. The tumorigenicity of these cell lines was assayed in nude mice. Although the series of cell lines studied provided a broad spectrum of LETS protein expression, both quantitatively and qualitatively, there does not appear to be a correlation between tumorigenicity and decreased expression of the LETS protein.In a series of transformed, nontumorigenic hybrids, the LETS protein expression was found to be altered with respect to both decreased organizational complexity and decreased content. These hybrids continue to express a number of other transformed phenotypes. Conversely, a number of tumorigenic hybrids continue to express relatively high levels of LETS protein when compared with nontumorigenic hybrids. Thus an alteration in LETS protein expression by itself, or in concert with a spectrum of other transformation properties, does not appear to be a sufficient requirement for tumorigenicity and lends further support to an apparent separate control of the transformed versus tumorigenic phenotype.     

Equivalent Circuit of Frog Atrial Tissue as Determined by Voltage Clamp-Unclamp Experiments

The equivalent circuit that has been used in the analysis of nerve voltage-clamp data is that of the membrane capacity in parallel with the membrane resistance. Voltage-clamp experiments on frog atrial tissue indicate that this circuit will not suffice for this cardiac tissue. The change in membrane current associated with a step change in membrane potential does not show a rapid spike of capacitive current as would be expected for the simple parallel resistance-capacitance network. Rather, there is a step change in current followed by an exponential decay in current with a time constant of about 1 msec. This relatively slow capacitive charging current suggests that there is a resistance in series with the membrane capacity. A possible equivalent circuit is that of a series resistance external to the parallel resistance-capacitance network of the cell membranes. Another possible circuit assumes that the series resistance is an integral part of the cell membrane. The data presented in this paper demonstrate that the equivalent circuit of a bundle of frog atrial muscle is that of an external resistance in series with the cell membranes.     

The widespread nature of phenotypic variability in hepatomas and cell lines, in the form of a geometric series

The phenomenon of geometric phenotypic variability is described and its widespread occurrence is established by a new analysis of data from a literature survey of quantitative variation in 39 different enzymes and other cell products in hepatomas and cell lines. The range of variation from hepatoma to hepatoma or from cell line to cell line was between 3- and 700-fold, depending on the particular cell product. By collating together and normalizing the data for the enzymes and other cell products surveyed, it was demonstrated in a statistically valid manner that the quantitative variation for most, if not all, of the enzymes and serum albumin was not random, but followed a geometric series, the consecutive terms of which differed by a factor of square root 2. In addition, examples are presented to show that quantitative inheritance in normal tissues also occurs along this geometric series.     

Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype

To clarify the mechanism underlying resistance to interferon (IFN) by the hepatitis C virus (HCV) in patients with chronic hepatitis, we attempted to develop an IFN-resistant HCV replicon from the IFN-sensitive 50-1 replicon established previously. By treating 50-1 replicon cells with a prolonged low-dose treatment of IFN-alpha and then transfecting the total RNA derived from the IFN-alpha-treated replicon cells, we successfully obtained four clones (named 1, 3, 4, and 5) of HCV replicon cells that survived against IFN-alpha (200 IU/ml). These cloned cells were further treated with IFN-alpha or IFN-beta (increased gradually to 2000 or 1000 IU/ml, respectively). This led to four replicon cell lines (alphaR series) possessing the IFN-alpha-resistant phenotype and four replicon cell lines (betaR series) possessing the IFN-beta-resistant phenotype. Furthermore, we obtained an additional replicon cell line (alphaRmix) possessing the IFN-alpha-resistant phenotype by two rounds of prolonged treatment with IFN-alpha and RNA transfection as mentioned above. Characterization of these obtained HCV replicon cell lines revealed that the betaR series were highly resistant to both IFN-alpha and IFN-beta, although the alphaR series containing alphaRmix were only partially resistant to both IFN-alpha and IFN-beta. Genetic analysis of these HCV replicons found one common amino acid substitution in the NS4B and several additional amino acid substitutions in the NS5A of the betaR series, suggesting that these genetic alterations are involved in the IFN resistance of these HCV replicons. These newly established HCV replicon cell lines possessing IFN-resistant phenotypes are the first useful tools for understanding the mechanisms by which HCV acquires IFN resistance in vivo.     

Synthesis and structure-activity relationship of aminopyrimidine IKK2 inhibitors

The synthesis and structure-activity relationship of a novel series of aminopyrimidines are exemplified. Results of key compounds from within this series in the E-selectin reporter cell assay are also reported.     

Studies on Stabilization Ponds for Domestic Sewage in India

For one year a single cell stabilization pond, T. T. Nagar, Bhopal (August, 1968 to July, 1969) and series stabilization ponds, Shahpur, Bhopal (January, 1969 to December, 1969) were studied. Climatological conditions in Bhopal were favourable for the treatment of sewage in stabilization ponds. There was a considerable reduction in BOD, total nitrogen, phosphate, coliforms and enterococci during the process of treatment. The reductions were highest in series ponds. Studies on diurnal variations showed that highest pH, dissolved oxygen, algal cell number and lowest alkalinity values occurred around 4.00 p.m. Algal flora present in the single cell pond consisted of 31 species representing 27 genera. In the series ponds 33 species belonging to 30 genera and 37 species belonging to 33 genera were recorded for the primary and secondary ponds, respectively.     

Synthesis and antitumour evaluation of novel 2-phenylbenzimidazoles

A new series of fluorinated and non-fluorinated 2-phenylbenzimidazoles bearing oxygenated substituents on the phenyl ring has been synthesized. Synthesis of the new series was based on our previous discovery of 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610) as a potent and selective antitumour agent in vitro (sub-nanomolar GI(50) in sensitive human cancer cell lines), but with poor aqueous solubility and lack of a definitive cellular target limiting further development. In this study we test the hypothesis that 2-phenylbenzimidazoles with similar substitution patterns to PMX 610 would retain potent antitumour activity but with potentially superior pharmaceutical properties. In general the new compounds were less active than the former benzothiazole series in vitro when tested against the breast cancer cell lines MCF-7 and MDA 468; however the two most active compounds in the present series (3j and 3k) exhibit low micromolar GI(50) values in both cell lines and provide the opportunity for further chemical derivatization with a view to target identification.     

Relationship between multidrug resistance,hypersensitivity to resistance modifiers and cell volume in Chinese hamster ovary cells.

We have previously shown that very high levels of hypersensitivity to several resistance modifiers are correlated with increasing multidrug resistance in a series of Chinese hamster ovary cell lines. We have now selected a new member of the series which is an exception to this correlation in that although it is almost twice as multidrug resistant as the cell line from which it was derived, it shows much less hypersensitivity to resistance modifiers. Level of resistance modifier hypersensitivity correlated with the level of reduction of verapamil accumulation in these cells, and with the density of P-glycoprotein, but since the selection of this cell line has involved a doubling of cell volume, it was not correlated with total amount of P-glycoprotein.     

Compensation mechanisms in hemopoietic stem cell pool (CFUs) under the conditions of experimental chronic gamma-irradiation

The kinetics, proliferation and differentiation potentials of hemopoietic stem cells (CFUs) of bone marrow and spleen were investigated in CBA-line mice in the early period (1-30 days) of chronic gamma-irradiation at a dose rate of 0.16 Gy/day to attain a cumulative dose of 4.8 Gy. The results of the experimental study showed the prevalent maintenance of productivity of granulocytic and erythrocytic hemopoietic cell series within the range of reference values, persistent inhibition of the megakaryocytic series (in terms of all hemopoiesis parameters of interest), more marked suppression of the population of polypotential CFUs in the bone marrow as compared with that in the spleen. The obtained results indicated that the mechanisms of hemopoiesis compensation at stem cell pool level were as follows: the increase in proliferation potency of erythrocytic and in polypotential precursors, the rise in the proportion of granulocytic precursors in the real differentiation potential of CFUs, and the processes of repopulation manifested with different intensity in all stem cell populations under study. For maintenance of the necessary productivity of CFUs in each of hemopoietic cell series, consecutively or simultaneously, several compensatory-adaptive mechanisms are started, which allows the avoidance of a sharp competition between hemopoietic cell series under the conditions of stem cell pool depopulation, and preservation of the hemopoiesis as a whole.     

Structure-activity relationship and liver microsome stability studies of pyrrole necroptosis inhibitors

Necroptosis is a regulated caspase-independent cell death pathway resulting in morphology reminiscent of passive non-regulated necrosis. Several diverse structure classes of necroptosis inhibitors have been reported to date, including a series of [1,2,3]thiadiazole benzylamide derivatives. However, initial evaluation of mouse liver microsome stability indicated that this series of compounds was rapidly degraded. A structure-activity relationship (SAR) study of the [1,2,3]thiadiazole benzylamide series revealed that increased mouse liver microsome stability and increased necroptosis inhibitory activity could be accomplished by replacement of the 4-cyclopropyl-[1,2,3]thiadiazole with a 5-cyano-1-methylpyrrole. In addition, the SAR and the cellular activity profiles, utilizing different cell types and necroptosis-inducing stimuli, of representative [1,2,3]thiadiazole and pyrrole derivatives were very similar suggesting that the two compound series inhibit necroptosis in the same manner.     

Synthesis and biological activity of ferrocenyl indeno[1,2-c]isoquinolines as topoisomerase II inhibitors

Three series of indeno[1,2-c]isoquinolines bearing a ferrocenyl entity were synthesized and evaluated for DNA interaction, topoisomerase I and II inhibition, and cytotoxicity against breast human cancer cell lines. In the first and second series, the ferrocenyl scaffold was inserted as a linker between the two nitrogen atoms. In the last series, it was introduced at the end of the carbon chain. The present study showed that the ferrocenyl entity enhanced the topoisomerase II inhibition. Most compounds showed a potent growth inhibitory effect on MDA-MB-231 cell line with the IC₅₀ in μM range.     

Nanos is required in somatic blast cell lineages in the posterior of a mollusk embryo

During animal development, blast cell lineages are generated by repeated divisions of a mother cell into a series of daughter cells, often with a specific series of distinct fates. Nanos is a translational regulator that is involved in germline development in diverse animals and also involved in somatic patterning in insects. Recently, Nanos was found to be required for maintenance of stem cell divisions in the Drosophila germline. We have found that in the mollusk Ilyanassa, Nanos messenger RNA and protein are specifically localized in the mesendodermal blast cell lineage derived from the strongly conserved 4d cell. Nanos activity is required for differentiation of multiple tissues that are derived from the 4d cell, showing that IoNanos is required for somatic development in this embryo. At the cellular level, we show that IoNanos activity is required for the highly stereotyped cleavage pattern of the 4d lineage, the proliferative capacity of the blast cells, and the marked asymmetry of the blast cell divisions. These results suggest that IoNanos is involved in regulating blast cell behaviors in the 4d lineage.     

Silent development of memory progenitor B cells

T cell-dependent immune responses generate long-lived plasma cells and memory B cells, both of which express hypermutated Ab genes. The relationship between these cell types is not entirely understood. Both appear to emanate from the germinal center reaction, but it is unclear whether memory cells evolve while obligatorily generating plasma cells by siblings under all circumstances. In the experiments we report, plasma cell development was functionally segregated from memory cell development by a series of closely spaced injections of Ag delivered during the period of germinal center development. The injection series elevated serum Ab of low affinity, supporting the idea that a strong Ag signal drives plasma cell development. At the same time, the injection series produced a distinct population of affinity/specificity matured memory B cells that were functionally silent, as manifested by an absence of corresponding serum Ab. These cells could be driven by a final booster injection to develop into Ab-forming cells. This recall response required that a rest period precede the final booster injection, but a pause of only 4 days was sufficient. Our results support a model of memory B cell development in which extensive affinity/specificity maturation can take place within a B cell clone under some circumstances in which a concomitant generation of Ab-forming cells by siblings does not take place.     

Graded amplification of the Na,K-ATPase across a subclonal series: effects on membrane physiology

We have generated a series of clonally related cell lines which differ in the level of amplified expression of the Na,K-ATPase. These lines, originally derived from the ouabain resistant HeLa variant C+, expressed different numbers of binding sites for the Na,K-ATPase inhibitor ouabain, ranging from 2.9 X 10(6)/cell to 11.8 X 10(6)/cell. Amplification of the genes for both subunits of the enzyme was also seen but was not strictly correlated with level of expression. The influxes of histidine and tetraphenylphosphonium were measured across a series, including HeLa S3 and revertants, expressing from 0.74 X 10(6) to 10.5 X 10(6) ouabain-binding sites per cell. Tetraphenylphosphonium influx rate, presumed to be a function of membrane potential, varied linearly with ouabain binding site number, while histidine influx varied with the log of ouabain binding site number. Our results suggest that membrane potential increases in a simple fashion across our series of amplified lines. However, histidine influx was unaffected by treatments which cause membrane depolarization and a decrease in tetraphenylphosphonium influx rate. We propose that increasing histidine influx rates across our amplified series reflects exchange acceleration of L system transport due to increased intracellular pools of L system reactive amino acids. The Na,K-ATPase is ultimately responsible for most active transport across the plasma membrane. The consistent, graded physiological alterations seen across this series of closely related lines, chosen for graded enzyme expression, demonstrate the value of this novel genetic approach to the study of the energization of membrane transport.     

Time series analysis supporting the hypothesis that enhanced cosmic radiation during germ cell formation can increase breast cancer mortality in germ cell cohorts

 Techniques from cancer epidemiology and time series analysis were used to explore the hypothesis that cosmic radiation can induce germ cell changes leading to increases in future breast cancer mortality. A birth cohort time series for female breast cancer mortality was obtained using a model-independent, age-period-cohort analysis on age-specific mortality data for 1940–1990. The birth cohort series contained several oscillatory components, which were isolated and compared to the corresponding frequency components of a cosmic ray surrogate time series – Greenland ice-core ¹⁰Be concentrations. A technique, referred to as component wave-train alignment, was used to show that the breast cancer and cosmic ray oscillations were phase-locked approx. 25 years before the time of birth. This is consistent with the time of germ cell formation, which occurs during the fetal development stage of the preceding generation. Evidence is presented that the observable oscillations in the birth cohort series were residues of oscillations of much larger amplitude in the germ cell cohort, which were attenuated by the effect of the broad maternal age distribution. It is predicted that a minimum of 50% of breast cancer risk is associated with germ cell damage by cosmic radiation (priming event), which leads to the development of individuals with a higher risk of breast cancer. It is proposed that the priming event, by preceding other steps of carcinogenesis, works in concert with risk factor exposure during life. The priming event is consistent with epigenetic changes such as imprinting. Received: 10 June 1996 / Revised: 4 December 1996 / Accepted: 19 December 1996     

CHLOROPHYLL-DEFICIENT CELL LINES WHICH ARE GENETICALLY UNCHARACTERIZED CAN BE INAPPROPRIATE FOR USE AS PHENOTYPIC MARKERS IN DEVELOPMENTAL STUDIES

Periclinal chloroplast chimeras are genetic mosaics which possess shoot apices composed of one or more chlorophyll-deficient histogens and can exist as a series of arrangements of normal and mutant layers (A-B-B, A-B-A, etc.). Three periclinal chimeral cultivars of Sansevieria trifasciata L., each of which possesses normal green cell layer(s) but a genetically different chlorophyll-deficient cell layer(s), were utilized to study the effect of genotype on the ability of the cell layers of leaf cuttings and of cultured leaf tissue to regenerate shoots. The epidermis and LI derivatives were apparently incapable of shoot regeneration via leaf cutting, yet in two cultivars produced some shoots in vitro. In two of the cultivars, the chlorophyll-deficient cells never produced shoots. In the third, the capability of chlorophyll-deficient cell layers to produce shoots was less in vitro than in vivo, indicating that when determining morphogenic potential, direct comparisons between in vitro and in vivo systems may not be valid. Results also demonstrate that because genetically different albino cell layers can differ in their morphogenic response, utilizing a series of periclinal chimeras is useful only if the series is composed of the same two genotypes.