Paclitaxel-induced apoptosis in HeLa cells is serum dependent

Exposure of HeLa cells to different concentrations of the antineoplastic drug paclitaxel resulted in a loss of cell viability that was dependent on the concentration and time of exposure to the drug. This phenomenon was associated with the appearance of nuclear morphology typical of apoptosis and DNA breakage into a “ladder” pattern of discrete fragments of nucleosomal size. The induction of cell death was dependent on the serum concentration of the culture media, repressed by pretreatment with a cAMP-dependent protein kinase (PKA) inhibitor, and enhanced by increasing the cell proliferation with previous exposure to a cAMP-analog and a protein kinase-C (PKC) inducer. The proliferative index modifies the effect of taxol on HeLa cells, probably by means of a more rapid accumulation of cells in the G2/M cycle blockage point, although a direct participation of PKA and PKC should not be excluded. © 1997 John Wiley & Sons, Inc.     

Cell cycle control by anchorage signaling

Virtually all the cells constituting solid organs in adult animals require anchorage to the extracellular matrix for their proliferation and survival. When deprived of anchorage, those cells arrest in G(1) phase of the cell cycle and die of apoptosis known as anoikis. However, if malignantly transformed, cells no longer require such an anchorage to proliferate and survive, and it is generally thought that the acquirement of this ability underlies the tumorigenic and metastatic capability of malignant cells. Therefore, for the past two decades, great efforts have been devoted to uncovering the nature of the anchorage signal and the mechanism by which this signal controls the G(1)-S transition in the cell cycle with little progress. However, several critical findings were recently made on anchorage signaling and the control of the cell cycle and cell death by this signaling. This review focuses on the newly emerging understanding and perspective of this highly important cell cycle and cell death regulation.     

Lamprey serum can kill HeLa and NB4 tumor cells

Nature consists primarily of living materials, many of which are pathogenic microorganisms capable of killing and converting other organisms into copies of themselves . For self-defense, eukaryotes have gradually evolved immune systems, including the innate and adaptive immune systems . Innate immunity has been found in plants, fungi, and metazoans; while jawed vertebrates have an evolved adaptive immune system in addition to the innate immunity defense . Agnathans, represented by lamprey and hagfish, are the oldest vertebrates that possess the adaptive immune defenses, although whether TCR and BCR exist in jawless vertebrates remains unknown.     

Growth of anchorage dependent mammalian cells on glycine-derivatized polystyrene in suspension culture

Summary Glycine-derivatized polystyrene beads were prepared and used as microcarriers to grow normal cells of human embryonic kidney, rhesus monkey kidney, and human foreskin fibroblasts in suspension cultures. Growth of the cells on polystyrene beads derivatized with other amino acids, peptides, and carboxylic acids also was investigated.     

Acid phosphatase of HeLa cells: properties and regulation of lysosomal activity by serum.

Although the subcellular distribution profile of acid phosphatase in HeLa cells is typical of a lysosomal enzyme, different lysosomal (70–80%) and supernatant forms (20–30%) have been demonstrated by their differences in pH activity curves, substrate specificities, thermal stability, sensitivity to inhibitors, and kinetics. Enzymes of the lysosomal fraction displayed anomalous kinetics in the hydrolysis of p-nitrophenyl phosphate. The major lysosomal acid phosphatase activity appears to be associated with the membrane.The total acid phosphatase activity in the cell is controlled by the concentration of serum in the medium. The specific activity in the homogenates of cells grown in high serum concentration (30%) is about twice that of cells grown in low serum concentration (1%). This doubling of specific activity holds for the lysosomal enzyme (or enzymes), but little change occurs in the supernatant form (or forms). Two other lysosomal enzymes, β-glucuronidase and N-acetyl-β-d-hexosaminidase, do not increase in specific activity. The serum-dependent formation of acid phosphatase is sensitive to cycloheximide, actinomycin D, and cordycepin. Cycloheximide blocks the increase in enzymatic activity immediately, whereas cordycepin and actinomycin D have no effect for at least 8 h. These findings suggest that de novo protein synthesis is involved in the induction of lysosomal acid phosphatase by serum and that the mRNA for this enzyme is relatively stable.     

Growth inhibition of capsaicin on HeLa cells is not mediated by intracellular calcium mobilization

The effects of capsaicin on cellular growth and intracellular calcium mobilization were examined in human cervical carcinoma derivation, HeLa cells. Capsaicin inhibited cellular growth and increased intracellular calcium level in HeLa cells. This capsaicin-induced intracellular calcium concentration rise was blocked by capsazepin, vanilloid (capsaicin) receptor antagonist. But, an intracellular calcium chelator BAPTA/AM did not block the inhibitory effect of capsaicin on cellular growth. These observations suggest that intracellular calcium mobilization is not required for the capsaicin-induced inhibition of cellular growth.     

Fibronectin is not a serum spreading factor for HeLa cells

The spreading of HeLa cells on plastic substratum is mediated by fibronectin-depleted foetal calf serum but not by fibronectin isolated by gelatin-Sepharose affinity chromatography. The same is true for freshly explanted chick embryonic chondrocytes. In contrast, BHK cell spreading exceeds 67% after 120 min at 37 degrees C in fibronectin-supplemented (10 micrograms ml-1) serum-free medium. Long-term cultivation of HeLa cells in Eagle's MEM supplemented with fibronectin-free serum is associated with the accumulation of cells in mitosis or before cytokinesis; many cells die and the remaining living cells, characterized by marked changes in morphology, multiply very slowly. It can be concluded therefore that fibronectin does not produce spreading in HeLa cells but forces them into mitosis.     

Studies on the control of ribosomal RNA synthesis in HeLa cells.

In many eucaryotic systems protein synthesis is coupled to ribosomal RNA synthesis such that shut-down of the former causes inhibition of the latter. We have investigated this stringency phenomenon in HeLa cells. The protein synthesis inhibitors cycloheximide and puromycin cause inactivation of both processes but valine starvation totally inhibits only the processing of 45-S RNA. DNA-dependent RNA polymerases from A, B and C (or I, II and III respectively) were extracted, separated partially by DEAE-cellulose chromatography and their activity levels determined. These do not decrease significantly during inhibition of protein synthesis. To find out whether or not form A is bound to its template under these conditions, proteins were removed from chromatin with the detergent sarkosyl. This does not affect bound RNA polymerase. Inhibition of protein synthesis caused up to 50% reduction in endogenous alpha-amanitin-insensitive chromatin-RNA-synthesising activity. This reduced level of activity was not affected by sarkosyl treatment. Levels in normal cells were stimulated. This result indicates that the form A RNA polymerase is not bound to its template when protein synthesis is inhibited.     

Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation

Bright nucleolar immunofluorescence was observed in HeLa S3 cells by immunostaining with a monoclonal antibody to the nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1). After 48 h of incubation in a serum-free medium, the nucleolar fluorescence was diminished and a general nuclear immunofluorescence was observed. This change in localization of fluorescence indicated that protein B23 had migrated out of the nucleoli. No gross morphological change in nucleoli was observed by light microscopy and the immunolocalization of another nucleolar phosphoprotein, C23, was unaffected by serum deprivation. Relocation of protein B23 in nucleoli was observed after refeeding with serum-containing medium. This re-entry process was not observed after treatment with actinomycin D (50 ng/ml-5 micrograms/ml), but the process was unaffected by cycloheximide (0.2 mM). Quantitation of protein B23 in the nucleoli of the control (fed) or starved HeLa cells was done by ELISA immunoassay. A marked decrease in the amount of protein B23 occurred in the nucleoli of the starved cells (11.8 micrograms B23/mgDNA) as compared with the control nucleoli (20.8 micrograms B23/mgDNA). The amount of protein B23 in the nucleoplasm (excluding nucleoli) was 70% higher in the starved cells. Protein B23 was analysed by one- and two-dimensional PAGE. Three components of protein B23 with slightly different molecular weights and pIs (37 kD/5.1, 35 kD/5.1 and 35 kD/5.3) were observed in nucleoli. The lower molecular weight components were predominantly found in the nucleoplasm.     

Growth control of normal and transformed cells

Both serum factors and protein synthesis are required for normal cell growth. Swiss 3T3 cells require the serum growth factors insulin and EGF (epidermal growth factor) during the initial part of the G₁ period, until they pass a restriction point about 2 h before the initiation of DNA synthesis. Concentration of cycloheximide that inhibit protein synthesis by as much as 70% dramatically lengthen the cell cycle before the restriction point, while the cell cycle after the restriction point remains nearly constant. These results are consistent with a model in which labile proteins are required for transit of cells past the serum-sensitive restriction point. The relation of these findings to the growth control of transformed cells is discussed.     

Regulation of acetyl-CoA carboxylase by glucagon in HeLa cells

A new method of purification of rat liver L-threonine deaminase has been developed, and the results obtained are compared with values obtained by other authors. Some properties of this enzyme (pH optimum, temperature optimum, thermal stability, specificity, etc.) have been examined and we found that the enzyme is inhibited by carbonate ions, that L-cysteine (a competitive inhibitor) is also an inactivator of the enzyme and that it is bound to the enzyme in a ratio of 0.25 mole of cysteine per mole of enzyme, supporting the hypothesis that the enzyme consists of 4 subunits.     

Influence of serum and poliovirus infection on the transport and accumulation of AIB in HeLa cells

The kinetics of active transport of the model amino acid AIB (aminoisobutyric acid) into HeLa cells were analysed. Serum withdrawal from cell-culture medium prior to transport assay strongly lowers the influx and steady-state distribution of AIB. Readdition of serum restores the rate of influx within 30 min, while the capacity to concentrate AIB is only partially reestablished. These changes are due to alterations in the activity of system A. In the time-course of poliovirus infection the initial rate of AIB transport shows two distinct changes. Early in the infection there is an increase in influx, followed by a strong decrease later in infection. These alterations are also attributed to transport system A.     

Disorganization of mitosis in HeLa cells by deuterium oxide

We assessed, by light and electron microscopy, the influence of deuterium oxide on the dynamics of mitosis and on the morphology of the mitotic apparatus in HeLa cells grown in vitro. A 2-h incubation of HeLa monolayers with low concentrations of D2O (1%-25%) in the medium increased the frequency of multipolar divisions up to 20 times the control level. Substitution of 10% and 25% D2O for H2O induced changes in the proportions of mitotic phases. These changes could be fully reversed to the control pattern after 1 h of recovery in non-deuterated medium. Fifty % D2O strongly inhibited, and 75% D2O blocked the cell cycle before prophase and at (pro-)metaphase. In cells treated with 50% D2O conspicuous morphological changes of the interphase chromatin as well as ultrastructural abnormalities of all mitotic phases were regularly observed. Overall, these results confirmed the antimitotic activity of deuterium oxide and revealed that it could also influence the cell cycle before mitosis. It is suggested that interference with diverse cellular constituents rather than a specific influence on microtubule turnover could be responsible for the disorganization of the cell cycle in HeLa cells by D2O.