Induction of quiescence by differentiating agents

The growth fraction of cancer cells, estimated by the monoclonal antibody Ki-67 labelling, and DNA content were determined simultaneously en K562 human leukemic cells by flow cytometry. Adriamycin, aclacinomycin A and fagaronine induced differentiation, as assessed by benzidine staining and glycophorin A expression. These drugs decreases the fraction of Ki-67 positive cells, Ki-67 negative cells displayed a G1, but also a G2 and a S DNA content in different proportions, indicating that induction of quiescent cells by differentiating agents is not a uniform process and is worthy of interest.     

Amino acids and their derivatives as radioprotective agents

Summary Numerous amino acids and their analogs are capable of protecting biological systems from the toxic effects of ionizing radiation. These radioprotective agents can be classified into two broad groups, depending upon the presence or absence of a free or potentially free sulfhydryl group. The sulfhydryl-containing compounds have been studied extensively and are thought to exert their radioprotective effects by several mechanisms, including free radical scavenging and hydrogen atom donation. Several non-sulfhydryl-containing amino acids are also being investigated for their radioprotective effects. These agents are less well known than the familiar sulfhydryl compounds, but possess very interesting protective qualities. In short, the study of amino acids and their derivatives as radioprotective agents continues to contribute to an understanding of processes involved in radiation toxicity and to offer new compounds with potential application to situations of human exposure.     

Induction of mouse liver glyoxalase I by hypobaric hypoxia

Hypobaric hypoxia at 0.45 atm induced a reversible increase of mouse liver glyoxalase I. The levels of this enzyme increased after an exposure of 20 h and 20 + 20 h, whereas the activity decreased to the control values after 20 h at room pressure. Before the treatment, some animals received tritiated leucine (i.p.). Glyoxalase I was purified to homogeneity. The pure enzyme from the treated animals showed 20-times more radioactivity than the controls. Thus, the increase in specific activity is due to new protein synthesized in response to the treatment at 0.45 atm. The activities of glyoxalase II and glutathione S-transferase were not affected by the treatment.     

Bone marrow osteoblast damage by chemotherapeutic agents

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-β1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-β1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.     

Induction of starfish oocyte maturation by disulfide-reducing agents

Oocyte maturation was found to be induced by disulfide-reducing agents such as dithiothreitol (DTT) and 2,3-dimercapto-1-propanol (BAL) in the starfish, Asterina pectinifera. The follicular envelopes around the oocytes broke and retracted into small clumps of cells on treatment with these reagents, as in the case of 1-methyladenine. Upon insemination, fertilizable eggs obtained by treatment with DTT formed a tight fertilization membrane and underwent cleavage. Such eggs developed normally to bipinnaria larvae. Cysteine and glutathione-SH had no effect in inducing oocyte maturation. On the other hand, pretreatment with sulfhydryl reagents such as p-chloromercurybenzoate (PCMB), iodoacetamide (IAM) and N-ethylmaleimide (NEM) completely suppressed 1-methyladenine-induced oocyte maturation. This inhibitory effect of sulfhydryl reagents on oocyte maturation was diminished by subsequent treatment with DTT or BAL with or without 1-methyladenine. Pretreatment with o-iodosobenzoate failed to inhibit 1-methyladenine-induced oocyte maturation.     

Induction of petite yeast mutants by membrane-active agents

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Reduced bone marrow pO2 following treatment with radioprotective drugs

The sensitizer adduct technique [( 3H]misonidazole binding) was used to assess the extent of murine bone marrow hypoxia following treatment with a variety of radioprotectors. The binding rates previously determined in vivo were compared to those obtained by incubating marrow cells in atmospheres of varying oxygen content. Parallel experiments demonstrated that the oxygen dependence of [3H]misonidazole binding (Km approximately 0.15% oxygen) was similar to the oxygen dependence of marrow radiosensitivity (Km approximately 0.2% oxygen). Maximally radioprotective doses of several drugs have been shown to increase the binding of [3H]misonidazole significantly in vivo. A comparison to the in vitro binding rates suggests that the average oxygen concentration in the marrow at times associated with radioprotection was on the order of 0.5 to 0.8% oxygen. The relative importance of marrow hypoxia to the overall radioprotective effects of different drugs may vary considerably. However, these results have demonstrated that certain radioprotective drugs can induce marrow hypoxia and this reduced pO2 may contribute to the efficacy of these agents.     

Induction of petite yeast mutants by membrane-active agents.

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Induction of morphological alterations by antineoplastic agents in yeast

Saccharomyces cerevisiae was used as an alternative experimental model in order to investigate the effects of antineoplastic agents on eukaryotic cells. After being exposed to the most common clinically used antineoplastic agents, yeast cells were examined under the light microscope. Folate and pyrimidine antagonists, platinum derivatives, mitomycin C, actinomycin D and bleomycin induced alterations in yeast cellular morphology, which were not observed following treatment with drugs belonging to any category other than the antineoplastics, leading to the suggestion that these alterations could potentially be used as an experimental tool in pre-screening for new chemotherapeutic leads.