Joyce M. Fisher

The influence of Cremophor EL on the cell cycle effects of paclitaxel (Taxol®) in human tumor cell lines

We have perfomed DNA flow analysis, mitotic index studies, time-lapse photography, and paclitaxel uptake studies of human tumor cell lines exposed to paclitaxel. DNA flow analysis demonstrated that cells began accumulating in G2/M within 6 hrs of exposure to paclitaxel; by 12 hrs over 50% of cells accumulated in G2/M at all concentrations tested. After 24 hrs of exposure to 10 nM paclitaxel, cells underwent non-uniform mitotic division resulting in multinucleated cells. Of cells treated with 30 nM to 1000 nM paclitaxel, 75% to 85% remained blocked in G2/M for up to 72 hrs. Although a large proportion of cells treated with higher concentrations of paclitaxel (10,000 nM) was blocked in G2/M, a significant proportion (10% to 40%) of these cells was also in Gl. Cells exposed to lower concentrations of paclitaxel (10 nM to 1000 nM) in medium containing 0.135% (v/v) Cremophor EL also had a relatively large proportion in Gl. Mitotic index studies demonstrated that the paclitaxel-induced G2/M block was initially a mitotic block and that cells remained in mitosis for up to 24 hrs. With additional time of exposure to paclitaxel, mitotic index and time-lapse studies indicated that cells attempted to complete mitosis; however, cytokinesis was inhibited and cells became multinucleated. Time-lapse photography revealed that paclitaxel markedly prolonged the time in mitosis from 0.5 hr to 15 hr. High levels of Cremophor EL (0.135% v/v) markedly reduced the number of cells in mitosis but did not alter the mitotic delay induced by paclitaxel.3H-paclitaxel uptake studies revealed that high concentrations of Cremophor EL did reduce the rate of uptake of paclitaxel into cells but had little effect on total paclitaxel accumulation. These results confirm that paclitaxel has striking effects on the cell cycle and show that high concentrations of Cremophor EL are capable of inducing a cell cycle block distinct from the mitotic block seen with paclitaxel. These results also demonstrate that cells exposed to paclitaxel for longer than 24 hours attempt to complete mitosis but the process of cytokinesis is inhibited. Together with cytotoxicity data, these results indicate that entry into and exit out of mitosis are prerequisites for paclitaxel cytotoxicity.

Changes in radiation survival curve parameters in human tumor and rodent cells exposed to paclitaxel (taxol

PURPOSE Late G2 and M are the most radiosensitive phases of the cell cycle. Cells exposed to paclitaxel develop a cell cycle arrest in G2/M. These studies were performed to assess the in vitro radiosensitization properties of paclitaxel in human tumor and rodent cell lines. METHODS AND MATERIALS The effect of paclitaxel on the radiation sensitivity of human breast (MCF-7), lung (A549), ovary (OVG-1) adenocarcinoma and Chinese hamster lung fibroblast V79 cells was determined with clonogenic assays. DNA flow cytometry studies were performed to define the cell cycle characteristics of the cells during irradiation. Survival curve parameters for all cell lines were determined with the use of a computer program which represents cell survival after radiation by a linear-quadratic model. RESULTS All cell lines developed a G2/M block after exposure to paclitaxel for 24 h. However, the degree of radiosensitization produced by paclitaxel varied among the cell lines. The maximal sensitizer enhancement ratio (SER) of paclitaxel was 1.8 in MCF-7 cells, 1.6 in OVG-1 cells, and 1.7 in V79 cells. However, no concentration of paclitaxel was able to enhance the radiation sensitivity of A549 cells. Paclitaxel increased the linear (alpha) component of the radiation survival curves in all cell lines. The quadratic (beta) component was unaffected by paclitaxel in the rodent cells. High concentrations of paclitaxel (> or = 1000 nM) increased beta slightly in the human cell lines but there was considerable variation in the effect of paclitaxel on beta. The cells which were sensitized to radiation by paclitaxel had a relatively small baseline alpha component, while A549 cells had a large alpha component. CONCLUSION We conclude that paclitaxel is a modest radiosensitizer in some, but not all, human tumor cells. Paclitaxel appears to cause radiosensitization mainly by increasing the alpha component of radiation survival curves. Cells that normally have a relatively small alpha component should exhibit the most radiosensitization in response to paclitaxel while cells with a large alpha component should show little or no radiosensitization after paclitaxel treatment. Because the greatest effect of paclitaxel is on the linear component of radiation survival curves, these results indicate that paclitaxel may be an effective radiation sensitizer in many human tumors treated at clinically relevant radiation doses of 2 Gy or less.

Characteristics of the inhibition of hemoglobin synthesis in rabbit reticulocytes by threo-α-amino-β-chlorobutyric acid

Abstract 1. 1. Addition of the valine antagonist, α-amino-β-chlorobutyric acid, to rabbit reticulocytes produces a rapid inhibition of hemoglobin synthesis with a subsequent increase in labelling of protein of the ribosomal fraction. 2. 2. At appropriate antagonist: valine ratios, the incorporation of valine itself occurs at the ribosomal level, but is inhibited in hemoglobin. This indicates that the block in hemoglobin synthesis is caused by substitution of the analogue for valine at specific valine sites, and it is suggested that the loci of these substitutions may be the Val-Val residues on the precursor protein of the β-chain. 3. 3. The antagonist also inhibits [ 14 C]isoleucine incorporation into hemoglobin, but unlike other amino acids, isoleucine does not accumulate in protein of the ribosomal fraction. This observation is discussed in relation to the characteristics of isoleucine incorporation into reticulocyte proteins and the location of residues of this amino acid near the NH 2 -terminal end of the α-chain of rabbit hemoglobin. 4. 4. Synthesis of both the α- and β-chains of hemoglobin is blocked by the antagonist and there is no evidence for the preferential synthesis and release of either chain. 5. 5. Abortive protein formed in the presence of the antagonist is rapidly degraded into trichloroaceeric acid-soluble products.

Hemin control of globin synthesis: Action of an inhibitor formed in the absence of hemin on the reticulocyte cell-free system and its reversal by a ribosomal factor

Abstract The inhibitor of globin synthesis formed by incubation of the ribosome-free supernatant fraction of rabbit reticulocytes may be partially purified by precipitation at pH 5. It is inactivated by incubation with trypsin and is therefore presumed to be a protein. Incubation of a cell-free incorporating system with inhibitor results in disaggregation of the polyribosomes, indicating a block at the site of initiation. There is a lag phase of about 2 min before any effect on the polyribosome profile becomes evident. This suggests either that this time is required for interaction between the inhibitor and a component of the hemoglobin-synthesizing system or that the pool of a component past the block must be depleted before the inhibition becomes evident. The inhibitor blocks the synthesis of the α- and β-chains of hemoglobin equally. It also inhibits the formation of non-hemoglobin proteins, although there is a 10-min delay before inhibition of synthesis of these proteins becomes evident. A factor isolated by KCl extraction of reticulocyte ribosomes and eluted with 0.15 M KCl from a DEAE-cellulose column can overcome the action of the inhibitor. This fraction is required for protein synthesis by a reconstituted system containing the KCl-extracted reticulocyte ribosomes.

Hemin control of globin synthesis: Effect of a translational repressor on Met-tRNAf binding to the small ribosomal subunit and its relation to the activity and availability of an initiation factor

Abstract The block in peptide chain initiation brought about by hemin deficiency of the rabbit reticulocyte lysate system is accompanied by a marked decrease in Met-tRNA f bound to the small ribosomal subunit. Under conditions of hemin supplementation, a similar reduction of Met-tRNA f binding is caused by the addition of the translational repressor which is formed by incubation of the ribosome-free supernatant fraction in the absence of hemin. Both types of inhibition can be reversed by an initiation factor obtained from reticulocyte ribosomes. The activity of a suboptimal concentration of initiation factor in promoting Met-tRNA f binding is incompletely inhibited by a large dose of repressor. In a partial system, where translation is blocked by cycloheximide, the repressor has no effect on Met-tRNA f binding promoted by added initiation factor. Cycloheximide increases the Met-tRNA f bound under conditions of hemin supplementation and also permits its slow accumulation under conditions of hemin deficiency. This is attributed to its action in blocking both translation and the resulting turnover of Met-tRNA f bound to the small ribosomal subunit. No change in the distribution of initiation factor between different ribosome particles was found under inhibited conditions. The results indicate that the hemin-controlled repressor has no direct effect on either the activity or the availability of the initiation factor.

Taxol in combination with doxorubicin or etoposide : possible antagonism in vitro

Background. Taxol is a novel chemotherapeutic agent that promotes microtubule assembly and stabilizes tubulin polymer formation. Clinical evaluation of its antineoplastic activity as a single agent and in combination with other chemotherapeutic drugs is in progress.

2,9-Dimethyl-1,10-phenanthroline (neocuproine): a potent, copper-dependent cytotoxin with anti-tumor activity

2,9-Dimethyl-1,10-phenanthroline (2,9-DMP), a copper-specific chelator, was a potent cytotoxin against L1210 cells in vitro; its activity was dependent upon available Cu2+ in the medium. Other divalent ions, Fe2+ and Zn2+, were ineffective as promoters of growth inhibition. As the copper chelate, a 4 microM solution produced a 4 log kill after a 1-hr incubation. This was in marked contrast to 1,10-phenanthroline, whose inhibition of cell growth was overcome by added Cu2+, Fe2+ and Zn2+. Cellular uptake of labeled 2,9-dimethyl-1,10-phenanthroline also required added Cu2+ in the medium. This transport was energy dependent, and the drug was concentrated over 200-fold by the cells. In preliminary evaluations, copper-2,9-DMP showed significant chemotherapeutic activity against the P388 murine lymphoma in vivo.

in vivo modulation of glutathione by buthionine sulfoximine: Effect on marrow response to melphalan☆

The effect of giving buthionine sulfoximine (BSO), 0.0265 g/mouse (6 mM), at 12 and 6 hr before treatment with melphalan--0.0 mg, 3 mg, 6 mg, and 9 mg/kg, was studied in C3H mice, and was compared with control groups that received normal saline 12 and 6 hr before identical melphalan treatment. BSO treatment resulted in depletion of GSH levels in bone marrow, liver, and muscle to 65, 13, and 41% of control levels, respectively. Hematological toxicity was assessed by measurement of CFU-S survival and peripheral white cell counts. CFU-S survival decreased with increasing doses of melphalan, but no difference was observed with BSO pre-treatment. Likewise, WBC counts following melphalan 9 mg/kg, were similar irrespective of BSO pre-treatment. These data suggest that the marrow toxicity seen with melphalan is not worsened by pre-treatment with BSO and that if tumors can be pre-sensitized with BSO, there may be a clinical role for melphalan/BSO drug combination.

Endogenous copper is cytotoxic to a lymphoma in primary culture which requires thiols for growth

With the use of bathocuproine sulfonate, a copper-specific chelator as an indicator, we have demonstrated that copper ions, present as a natural medium constituent are toxic to the growth of a lymphoma in primary culture and are principally responsible for the growth requirement of mercaptoethanol and other thiols. By chelating trace copper normally present in the medium, bathocuproine sulfonate retarded the oxidation of cysteine to poorly utilized cystine, thus permitting its direct utilization by the cells for growth.

Showdomycin and its reactive moiety, maleimide: A comparison in selective toxicity and mechanism of action in vitro☆

Abstract Showdomycin, a C-nucleoside antibiotic, was twice as toxic to L1210 murine leukemia cells as to murine bone narrow progenitor cells of the granulocyte-macrophage series. Its aglycone, maleimide, was equally toxic to both cell lines. Cysteine, adenosine and the potent nucleoside transport inhibitor 6-[(2-hydroxy-5-nitrobenzyl)thio]-9-β- d -ribofuranosylpurine (HNBMPR) reversed the early stages of toxicity of showdomycin to L1210 cells, but did not reduce the toxicity of maleimide. At cytotoxic concentrations, showdomycin progressively inactivated the nucleoside uptake system to completion. This inhibition of nucleoside uptake was reversed by cysteine under conditions where it reversed cytotoxicity. The binding of 6-[(4-nitrobenzyl)thio]-9-β- d -ribofuranosylpurine (NBMPR) by L1210 cells was also inhibited by showdomycin, indicating that the antibiotic inactivated the nucleoside transport site. The data suggest that the C-nucleoside structure confers some selectivity to the cytotoxic action of maleimide, directing it toward the nucleoside transport system of the tumor cell.