Loss Of Colony-forming Ability Research Articles

Computational Discovery and Validation of NAD+ Biosynthesis As Unique Vulnerability in B-Lymphoid Malignancies

Background and Significance: Glucocorticoids have been a mainstay in virtually all therapy regimens for B-lymphoid malignancies since the 1950s. Glucocorticoids selectively kill B-cell leukemia and lymphoma cells but have very limited effects on myeloid and epithelial cells. Likewise, inhibitors of B-cell signaling (e.g. ibrutinib) and selective depletion of B-cells (e.g. rituximab, CD19 CAR-T cells) have achieved important progress and are well tolerated. Drug discovery tool for selective targeting of B-cell malignancies: For these reasons, we developed an interactive computational tool ( lymphoblasts.org) to identify novel B-cell selective vulnerabilities based on integrated genetic and pharmacological compound screens ( Figure 1). Compound screening data from CTD and GDSC databases were reprocessed by fitting 4-parameter log-logistic dose response curves. Differential gene-dependency was also tested using DEMETER2 and Chronos scores, from Project Achilles RNAi and CRISPR datasets, respectively. To prioritize cell-type specific targets, compounds were ranked by average differential rank percentile of both compound-sensitivity and target-gene dependency. NAD+ biosynthesis as unique B-lymphoid vulnerability: Integration of genetic RNAi and CRISPR-based sensitivity data identified NMNAT1 and NAMPT as top-ranking B-cell selective targets that regulate NAD+ salvage and biosynthesis in the nucleus ( Figure 1). NAMPT, was previously identified as “pre-B-cell colony enhancing factor” and functions as rate-limiting enzyme in the salvage biosynthesis pathway of NAD+, which is critical for cellular energy metabolism. These genetic results were further corroborated by the identification of four small molecule inhibitors of NAMPT, with a striking B-cell-selective sensitivity profile. Compared to a panel of 1,078 myeloid leukemia and solid tumor cell lines, B-cell leukemia and lymphoma cell lines (n=98) were dramatically more sensitive to NAMPT inhibitors, including STF31, CAY10618, GMX1778 and Daporinad ( Figure 1). To verify these results based on the DepMap dataset and our drug discovery tool (lymphoblasts.org), we studied the effects of seven NAMPT inhibitors (CAY10618, FK866, GMX1778, OT82, STF118804, STF31 and KPT9274) on a panel of myeloid leukemia and solid tumor cell lines as well as PDX from patients with B-ALL and mantle cell lymphoma. Interestingly, three of these NAMPT inhibitors have demonstrated favorable PK/PD and safety profiles (NCT04281420, NCT04914845, NCT03921879). Genetic modeling of NAMPT-deficiency in B-cell malignancies. To model consequences of Nampt deletion in B-ALL, we transformed pre-B cells from Namptfl/fl mice with BCR-ABL1 and NRAS G12D oncogenes. Deletion of Nampt subverted competitive fitness of B-ALL cells, loss of colony-forming ability and cell cycle arrest. Additionally, we developed a conditional mouse model ( Namptfl/fl x Mb1-Cre) for B-cell-specific Nampt deletion, which resulted near-complete ablation of B-lymphopoiesis. Moreover, in vitro inducible deletion of Nampt caused rapid depletion and reduced colony formation in cultured bone marrow pre-B cells. Deletion of Nampt profoundly affected B-cell energy metabolism, significantly reduced FCCP-stimulated OCR and resulted in cell death owing to energy stress. Metabolomic studies of genetic Nampt-deletion using mass spectrometry revealed depletion of metabolites involved in amino acid metabolism, purine/pyrimidine metabolism, and the TCA cycle. Notably, phosphoserine was among the top-ranked downregulated metabolites, suggesting the involvement of one carbon pathway disruption in mediating energy crisis upon Nampt-deletion in B-ALL cells. Conclusions: These findings highlight that the classical ‘glucocorticoid paradigm’ can be extended to discover novel vulnerabilities, including NAD+ biosynthesis. Computational integration of genetic and pharmacological compound screens was particularly powerful in identifying previously unrecognized opportunities to leverage selective vulnerabilities of B-lymphoid leukemia and lymphoma. Our genetic studies corroborate the unique role of NAMPT and NMNAT1 in B-cell development and metabolic regulation. Further studies will focus on efforts to repurpose three clinically approved NAMPT small molecule inhibitors for the treatment of refractory B-cell malignancies.

The phospholipid-repair system LplT/Aas in Gram-negative bacteria protects the bacterial membrane envelope from host phospholipase A2 attack

Secretory phospholipases A2 (sPLA2s) are potent components of mammalian innate-immunity antibacterial mechanisms. sPLA2 enzymes attack bacteria by hydrolyzing bacterial membrane phospholipids, causing membrane disorganization and cell lysis. However, most Gram-negative bacteria are naturally resistant to sPLA2 Here we report a novel resistance mechanism to mammalian sPLA2 in Escherichia coli, mediated by a phospholipid repair system consisting of the lysophospholipid transporter LplT and the acyltransferase Aas in the cytoplasmic membrane. Mutation of the lplT or aas gene abolished bacterial lysophospholipid acylation activity and drastically increased bacterial susceptibility to the combined actions of inflammatory fluid components and sPLA2, resulting in bulk phospholipid degradation and loss of colony-forming ability. sPLA2-mediated hydrolysis of the three major bacterial phospholipids exhibited distinctive kinetics and deacylation of cardiolipin to its monoacyl-derivative closely paralleled bacterial death. Characterization of the membrane envelope in lplT- or aas-knockout mutant bacteria revealed reduced membrane packing and disruption of lipid asymmetry with more phosphatidylethanolamine present in the outer leaflet of the outer membrane. Moreover, modest accumulation of lysophospholipids in these mutant bacteria destabilized the inner membrane and rendered outer membrane-depleted spheroplasts much more sensitive to sPLA2 These findings indicated that LplT/Aas inactivation perturbs both the outer and inner membranes by bypassing bacterial membrane maintenance mechanisms to trigger specific interfacial activation of sPLA2 We conclude that the LplT/Aas system is important for maintaining the integrity of the membrane envelope in Gram-negative bacteria. Our insights may help inform new therapeutic strategies to enhance host sPLA2 antimicrobial activity.

The Chromosomal parDE2 Toxin-Antitoxin System of Mycobacterium tuberculosis H37Rv: Genetic and Functional Characterization.

Mycobacterium tuberculosis H37Rv escapes host-generated stresses by entering a dormant persistent state. Activation of toxin-antitoxin modules is one of the mechanisms known to trigger such a state with low metabolic activity. M. tuberculosis harbors a large number of TA systems mostly located within discernible genomic islands. We have investigated the parDE2 operon of M. tuberculosis H37Rv encoding MParE2 toxin and MParD2 antitoxin proteins. The parDE2 locus was transcriptionally active from growth phase till late stationary phase in M. tuberculosis. A functional promoter located upstream of parD2 GTG start-site was identified by 5′-RACE and lacZ reporter assay. The MParD2 protein transcriptionally regulated the PparDE2 promoter by interacting through Arg16 and Ser15 residues located in the N-terminus. In Escherichia coli, ectopic expression of MParE2 inhibited growth in early stages, with a drastic reduction in colony forming units. Live-dead analysis revealed that the reduction was not due to cell death alone but due to formation of viable but non-culturable cells (VBNCs) also. The toxic activity of the protein, identified in the C-terminal residues Glu98 and Arg102, was neutralized by the antitoxin MParD2, both in vivo and in vitro. MParE2 inhibited mycobacterial DNA gyrase and interacted with the GyrB subunit without affecting its ATPase activity. Introduction of parE2 gene in the heterologous M. smegmatis host prevented growth and colony formation by the transformed cells. An M. smegmatis strain containing the parDE2 operon also switched to a non-culturable phenotype in response to oxidative stress. Loss in colony-forming ability of a major part of the MParE2 expressing cells suggests its potential role in dormancy, a cellular strategy for adaptation to environmental stresses. Our study has laid the foundation for future investigations to explore the physiological significance of parDE2 operon in mycobacterial pathogenesis.

Inhibition of phosphatidylinositol 3-kinase/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in a caspase-dependent manner through mitochondrial dysfunction and DNA damage response in established and primary cultured glioblastoma cells.

Identification of therapeutic strategies that might enhance the efficacy of B-cell lymphoma-2 (Bcl-2) inhibitor ABT-737 [N-{4-[4-(4-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide] is of great interest in many cancers, including glioma. Our recent study suggested that Akt is a crucial mediator of apoptosis sensitivity in response to ABT-737 in glioma cell lines. Inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt are currently being assessed clinically in patients with glioma. Because PI3K/Akt inhibition would be expected to have many proapoptotic effects, we hypothesized that there may be unique synergy between PI3K inhibitors and Bcl-2 homology 3 mimetics. Toward this end, we assessed the combination of the PI3K/Akt inhibitor NVP-BKM120 [5-(2,6-dimorpholinopyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amine] and the Bcl-2 family inhibitor ABT-737 in established and primary cultured glioma cells. We found that the combined treatment with these agents led to a significant activation of caspase-8 and -3, PARP, and cell death, irrespective of PTEN status. The enhanced lethality observed with this combination also appears dependent on the loss of mitochondrial membrane potential and release of cytochrome c, smac/DIABLO, and apoptosis-inducing factor to the cytosol. Further study revealed that the upregulation of Noxa, truncation of Bid, and activation of Bax and Bak caused by these inhibitors were the key factors for the synergy. In addition, we demonstrated the release of proapoptotic proteins Bim and Bak from Mcl-1. We found defects in chromosome segregation leading to multinuclear cells and loss of colony-forming ability, suggesting the potential use of NVP-BKM120 as a promising agent to improve the anticancer activities of ABT-737.

Ionizing radiation-induced responses in human cells with differing TP53 status.

Ionizing radiation triggers diverse responses in human cells encompassing apoptosis, necrosis, stress-induced premature senescence (SIPS), autophagy, and endopolyploidy (e.g., multinucleation). Most of these responses result in loss of colony-forming ability in the clonogenic survival assay. However, not all modes of so-called clonogenic cell “death” are necessarily advantageous for therapeutic outcome in cancer radiotherapy. For example, the crosstalk between SIPS and autophagy is considered to influence the capacity of the tumor cells to maintain a prolonged state of growth inhibition that unfortunately can be succeeded by tumor regrowth and disease recurrence. Likewise, endopolyploid giant cells are able to segregate into near diploid descendants that continue mitotic activities. Herein we review the current knowledge on the roles that the p53 and p21WAF1 tumor suppressors play in determining the fate of human fibroblasts (normal and Li-Fraumeni syndrome) and solid tumor-derived cells after exposure to ionizing radiation. In addition, we discuss the important role of WIP1, a p53-regulated oncogene, in the temporal regulation of the DNA damage response and its contribution to p53 dynamics post-irradiation. This article highlights the complexity of the DNA damage response and provides an impetus for rethinking the nature of cancer cell resistance to therapeutic agents.

Similar Extent of Apoptosis Induction at Doses of X-Rays and Neutrons Isoeffective for Cell Inactivation

The relative biological effectiveness (RBE) of neutrons differs for various biological endpoints, and for various cell and tissue types. With respect to the apoptosis induction, a whole range of values can be found in the literature, but the decisive factors are not clear. Most previous studies have used apoptosis-prone hematopoietic cells, whereas tumor cells have received little attention. The authors therefore decided to investigate apoptosis induction caused by X-rays and neutrons in a line of human melanoma cells, at doses which are isoeffective for the loss of colony-forming ability. Human melanoma cells Be11, expressing p53 wild-type protein, were used throughout. Exponentially growing cells were exposed to two pairs of isoeffective doses (at surviving levels 10% and 1%) of 240-kV X-rays and 5.8-MeV neutrons. 1-8 days after irradiation, the frequency of apoptosis in adherent cells was assessed by two-parameter flow cytometric analysis with a DNA-dye-exclusion annexin-V-binding assay as well as by morphological examination with DAPI staining. Apoptosis was induced most significantly 6-7 days after irradiation. The time courses, as well as the magnitudes of apoptosis induction, after isoeffective doses of X-rays and neutrons with respect to loss of colony-forming ability appeared to be comparable. RBE values in the range of 4-5 were estimated for apoptosis 4-8 days after irradiation by both the annexin V assay and morphological examination. Radiation-induced apoptosis depends on ionization density in the same way as cell inactivation in general does, i.e., the RBE is similar, and the ratio of cells dying by apoptosis to cells dying otherwise does not depend on radiation quality.

RNase E Maintenance of Proper FtsZ/FtsA Ratio Required for Nonfilamentous Growth of Escherichia coli Cells but Not for Colony-Forming Ability

Inactivation or deletion of the RNase E-encoding rne gene of Escherichia coli results in the growth of bacterial cells as filamentous chains in liquid culture (K. Goldblum and D. Apirion, J. Bacteriol. 146:128-132, 1981) and the loss of colony-forming ability (CFA) on solid media. RNase E dysfunction is also associated with abnormal processing of ftsQAZ transcripts (K. Cam, G. Rome, H. M. Krisch, and J.-P. Bouché, Nucleic Acids Res. 24:3065-3070, 1996), which encode proteins having a central role in septum formation during cell division. We show here that RNase E regulates the relative abundances of FtsZ and FtsA proteins and that RNase E depletion results in decreased FtsZ, increased FtsA, and consequently an altered FtsZ/FtsA ratio. However, while restoration of the level of FtsZ to normal in rne null mutant bacteria reverses the filamentation phenotype, it does not restore CFA. Conversely, overexpression of a related RNase, RNase G, in rne-deleted bacteria restores CFA, as previously reported, without affecting FtsZ abundance. Our results demonstrate that RNase E activity is required to maintain a proper cellular ratio of the FtsZ and FtsA proteins in E. coli but that FtsZ deficiency does not account for the nonviability of cells lacking RNase E.

Compaction of the Escherichia coli nucleoid caused by Cyt1Aa.

Compaction of the Escherichia coli nucleoid in the cell's centre was associated with the loss of colony-forming ability; these effects were caused by induction of Cyt1Aa, the cytotoxic 27 kDa protein from Bacillus thuringiensis subsp. israelensis. Cyt1Aa-affected compaction of the nucleoids was delayed but eventually more intense than compaction caused by chloramphenicol. The possibility that small, compact nucleoids in Cyt1Aa-expressing cells resulted in DNA replication run-out and segregation following cell division was ruled out by measuring relative nucleoid length. Treatments with membrane-perforating substances other than Cyt1Aa did not cause such compaction of the nucleoids, but rather the nucleoids overexpanded to occupy nearly all of the cell volume. These findings support the suggestion that, in addition to its perforating ability, Cyt1Aa causes specific disruption of nucleoid associations with the cytoplasmic membrane. In situ immunofluorescence labelling with Alexa did not demonstrate a great amount of Cyt1Aa associated with the membrane. Clear separation between Alexa-labelled Cyt1Aa and 4',6-diamidino-2-phenylindole (DAPI)-stained DNA indicates that the nucleoid does not bind Cyt1Aa. Around 2 h after induction, nucleoids in Cyt1Aa-expressing cells started to decompact and expanded to fill the whole cell volume, most likely due to partial cell lysis without massive peptidoglycan destruction.

Evidence against apoptosis as a major mechanism for reproductive cell death following treatment of cell lines with anti-cancer drugs.

An increase in apoptotic cells may be observed after treatment with chemotherapy, and many authors have assumed that anti-cancer drugs kill cells by inducing apoptosis. The most relevant endpoint of cell death following treatment of tumour cells is loss of reproductive ability as measured by a colony-forming assay, since cells with limited reproductive potential cannot regenerate a tumour. We have therefore investigated the relationship between apoptosis and reproductive cell death following in vitro treatment of mammalian cell lines with anti-cancer drugs. Markers of apoptosis (DNA ladders, TUNEL assay) were evaluated at various times after treatment of Chinese Hamster Ovary (CHO) cells, human bladder cancer MGH-U1 cells, and a murine T-lymphocytic cell line (CTLL-2) with several anti-cancer drugs. These markers were found infrequently, despite the use of doses that cause loss of colony-forming ability, except in CTLL-2 cells. We also transfected and expressed the human pro-apoptotic gene bax and the anti-apoptotic gene bcl-2 in MGH-U1 cells and compared cell survival after drug treatment with that of control cells transfected with the vector alone. Expression of these genes had at most small effects to influence cell survival. We conclude that apoptotic mechanisms had at most a minor role in leading to reproductive death of MGH-U1 and CHO cells after chemotherapy. When apoptosis is observed following treatment with anti-cancer drugs it may be a secondary event which occurs in lethally-damaged cells, leading to their lysis, rather than a primary event that leads to loss of reproductive integrity. © 2001 Cancer Research Campaign http://www.bjcancer.com

Precise excision of bacteriophage Mu DNA

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.

The Case Against Apoptosis

Apoptosis is not as dominant a process in cell loss from normal tissues and tumours as has sometimes been claimed. The term 'programmed cell death', which many authors regard as synonymous with apoptosis, is an unsatisfactory term that is best avoided. In studies on the response of tumours to drug and radiation treatment, the use of apoptosis assays concentrates attention on the first decade of cell killing (0–90%), whereas the outcome of treatment depends on multi-log cell kill; an assay of clonogenic cell survival is the appropriate method for this purpose. Loss of colony-forming ability is the key event in treated tumour cells, and the appearance of morphological and molecular evidence of apoptosis is probably downstream from this event. Published studies that have compared apoptosis and cell survival responses in tumour cells have generally failed to find a causal relationship.

Apoptosis and cytokine release induced by ionizing or ultraviolet B radiation in primary and immortalized human keratinocytes

We have compared the induction of apoptosis and cytokine release by UVB and gamma-radiation in primary (untransformed) and in two immortalized human epithelial/keratinocyte cell lines, HaCaT and KB (KB is now known to be a subline of the ubiquitous keratin-forming tumour cell line HeLa and we therefore designate it HeLa-KB). In both the primary and the immortalized cell lines apoptosis and release of the inflammatory cytokine interleukin-6 are induced rapidly following UVB irradiation. In contrast, only the immortalized cells undergo apoptosis and release interleukin-6 after gamma-irradiation and here the onset of apoptosis and cytokine release are delayed. The same distinction between primary and immortalized cells was observed when double-strand breaks were induced with the anticancer drug mitoxantrone, which stabilizes topoisomerase II-cleavable complexes. We suggest that immortalization may sensitize keratinocytes to the apoptogenic effect of ionizing radiation or mitoxantrone by deregulating normal cell cycle checkpoints. In both human keratinocytes and fibroblasts, cell killing, as assayed by loss of colony-forming ability, is not coupled to apoptosis. Immortalization increases resistance to gamma-radiation killing but sensitizes to apoptosis. In contrast, although immortalization also sensitizes to UVB-induced apoptosis, it does not affect UVB-induced cell killing. Apoptosis unambiguously indicates death at the single cell level but clonal cell survival integrates all the cellular and genetic processes which prevent or permit a scorable clone to develop.

Effects of cis-diamminedichloroplatinum(II) on Escherichia coli and bacteriophage systems.

The effects of cis-diamminedichloroplatinum(II) (cisplatin) on Escherichia coli cells and bacteriophages were investigated. The bacteriocidal effect of cisplatin was stronger on uvrA or recA mutants than on wild type cells. The drug, like UV, induced prophage development in lysogenic bacteria. Host cell reactivation of alpha3 replicative form (RF) I DNA treated with cisplatin in vitro was more efficient in wild type or recA cells than in uvrA host. When wild type cells were exposed to cisplatin, decay of the host's capacity to sustain the viral multiplication proceeded nearly in parallel with the loss of colony-forming ability, whereas the capacity of uvrA mutant was much more resistant to the drug, as compared with the viability. In the DNA preparation from cisplatin-treated alpha3-infected wild type cells, RF II was deficient, but the RF I molecules extracted from the cells were moderately infective. The microvirid gene A protein, required for RF I-->RF II conversion, was hardly detectable in wild type cells exposed to cisplatin. The possible relationship between uvr+-dependent repair and synthesis of the viral protein is discussed.

Clonogenic Inactivation of Colon Cancer-derived Cells Treated with 5-Fluorouracil and Indomethacin in Hybrid Spheroids

The clonogenic hybrid spheroid assay has been used to determine the toxicity of 5-fluorouracil (5-FU), alone or in combination with indomethacin, in LoVo cells (a human colon adenocarcinoma line). The principal finding was that 5-FU toxicity, determined as loss of colony-forming ability, increased as a function of dose (concentration x duration of exposure), and that indomethacin causes a generalized alleviation of 5-FU toxicity, but only if given concurrently with 5-FU. The implications of these findings in the control of cancer cells by 5-FU are discussed.

Dependence of Induction of Interphase Death of Chinese Hamster Ovary Cells Exposed to Accelerated Heavy Ions on Linear Energy Transfer

Induction of interphase death was examined in Chinese hamster ovary cells exposed to accelerated heavy ions (carbon, neon, argon and iron) of various linear energy transfers (LETs) (10-2000 keV/microm). The fraction of cells that underwent interphase death was determined by observing individual cells with time-lapse photography (direct method) as well as by counting cells undergoing interphase death made visible by the addition of caffeine (indirect method). After exposure to X rays, interphase death increased linearly with dose above a threshold of about 10 Gy, whereas it increased at a higher rate without a threshold after exposure to high-LET heavy ions. The relative biological effectiveness (RBE) compared to X rays, as determined at the 50% level of induction, increased with LET, reached a maximum at an LET of approximately 230 keV/microm and then decreased with further increase in LET. The range of LET values corresponding to the maximum RBE appears to be narrower for interphase death than for reproductive death (120-230 keV/microm), as assayed using loss of colony-forming ability as a criterion. The inactivation cross section for interphase cell death reached a plateau of 5-10 microm2. This means that the probability for the induction of interphase death by traversal of a single heavy-ion track through the nucleus (size: about 130 microm2) is about 0.04-0.08.

Enhancement of adriamycin toxicity by iron chelates is not a free radical mechanism.

The possible involvement of metal ions and free radicals in the cytotoxic mechanism of Adriamycin (ADR) was investigated, using a model system of Escherichia coli cells. It is shown that E. coli mediated the production of free radicals under anaerobic (ADR-semiquinone) and aerobic (superoxide) conditions. ADR-induced loss of colony-forming ability was enhanced by the addition of iron (Fe) chelates. These observations suggested that a Fenton-type free radical mechanism was responsible for ADR toxicity. However, the mortality rate was essentially unchanged by the exclusion of oxygen. It was also unaffected by the addition of H2O2, catalase, or chelating agents. Cu(II), Zn(II) or Mg(II) had no effect on ADR toxicity. ADR and iron chelates did not induce measurable amounts of DNA strand-breaks. These observations suggest a mechanism of ADR-induced cell killing that is enhanced by Fe chelates, but does not directly involve oxygen-derived free radicals.

Role of Fenton Chemistry in Thiol-Induced Toxicity and Apoptosis

Under certain conditions, many radioprotective thiols can be toxic, causing loss of colony-forming ability in cultured mammalian cells in a biphasic fashion whereby the thiols are not toxic at high or low concentrations of the drug, but cause decreased clonogenicity at intermediate (0.2-1.0 mM) drug levels. This symposium paper summarizes our studies using dithiothreitol (DTT) as a model thiol to demonstrate the role of Fenton chemistry in thiol toxicity. The toxicity of DTT in V79 cells has several characteristics: it is dependent on the medium used during exposure of cells to the drug; the toxicity is decreased or prevented by addition of catalase exogenously, but superoxide dismutase has no effect; the toxicity is increased by addition of copper, either free or derived from ceruloplasmin in serum; and the toxicity can be modified intracellularly by altering glucose availability or pentose cycle activity. Thus the data are consistent with a mechanism whereby DTT oxidation produces H2O2 in a reaction catalyzed by metals, predominantly copper, followed by reaction of H2O2 in a metal-catalyzed Fenton reaction to produce the ultimate toxic species, .OH. Studies comparing 12 thiols have shown that the magnitude of cell killing and pattern of dependence on thiol concentration vary among the different agents, with the toxicity depending on the interplay between the rates of two reactions: thiol oxidation and the reaction between the thiol and the H2O2 produced during the thiol oxidation. The addition of other metals, e.g. Zn2+, and metal chelators, e.g. EDTA, can also alter DTT toxicity by altering the rates of thiol oxidation or the Fenton reaction. Recent studies have shown that in certain cell lines thiols can also cause apoptosis in a biphasic pattern, with little apoptosis at low or high drug concentrations but greatly increased apoptosis levels at intermediate (approximately 3 mM) thiol concentrations. There appears to be a good correlation between those thiols that cause loss of clonogenicity and those that induce apoptosis, suggesting similar mechanisms may be involved in both end points. However, thiol-induced apoptosis is not prevented by addition of exogenous catalase. These observations are discussed in relation to the possible role of Fenton chemistry in induction of apoptosis by thiols.

Inactivation of mecA prevents recovery from the competent state and interferes with cell division and the partitioning of nucleoids in Bacillus subtilis.

The development of genetic competence in Bacillus subtilis requires the synthesis of ComK, a transcription factor, which is normally produced as a culture enters the stationary phase. This synthesis is known to be regulated in part by the protein MecA. Loss-of-function mutations in mecA result in overexpression of ComK and its appearance early during exponential growth. We show here that mecA inactivation also causes a loss of colony-forming ability, especially during stationary phase. This loss is accompanied by the appearance of cells in which normal nucleoid separation has failed to occur. Renografin gradient fractionation of mecA cultures grown to competence reveals that nearly 100% of the cells band at the low buoyant density characteristic of competent cells, and that this low density is competence-related. The loss of viability, the low buoyant density and the nucleoid separation defect, are all comK-dependent. The loss of viability can be reversed by even the transient introduction of mecA+. It is proposed that these effects of ComK overexpression are related to the DNA replication arrest normally exhibited by the competent cell fraction and that MecA is needed to reverse this arrest and to permit escape from the competent state. The shift of nearly 100% of the cells to light buoyant density in a mecA mutant culture strongly suggests that the MecA protein is a regulator of the cell-type-specific expression of competence.

Micronucleus induction and reproductive death in a human cell line exposed to low-energy argon beam

Little is known about the action of charged particles of very high linear energy transfer (LET) on human cells and, in particular, the relationship between DNA damage and reproductive death. The aim of this study was to measure the biological efficiency of a low-energy argon beam (E = 7.1 MeV/nucleon, LET = 1590 keV/micron) produced at GSI, Darmstadt, on a human melanoma cell line (CAL4) established in our Institute. Two different methods were used: the micronucleus (MN) test and the colony-forming assay. The MN test, using the cytochalasin-block method, is a measure of genotoxic damage. MN are scored in binucleate cells (BNC) and are formed from acentric fragments or whole chromosomes that have not been incorporated into daughter nuclei at mitosis. The colony-forming assay quantifies reproductive death. Parallel experiments were run with cobalt gamma-rays for comparison. After Co irradiation, the MN-free BNC dose-response curve coincided with that of the loss of colony-forming ability, suggesting the potential of the former as a predictive test of cell killing. After Ar irradiation, there was a dissociation between the two effects, especially at high doses: cell death was greater than the frequency of BNC with MN. The inactivation cross-section was 74 microns2; it was 39 microns2 for MN yield. Therefore, the relative biological effectiveness (RBE) was higher for cell killing than for MN yield (0.8 and 0.5, respectively at a Co dose of 3 Gy). The total MN count in BNC followed the same pattern of response as the fraction of BNC with MN.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.

Basonuclin is a protein possessing three pairs of zinc fingers and a nuclear localization signal. Expression of the gene is largely confined to keratinocytes of stratified squamous epithelia and hair follicles. In the epidermis and in stratified epidermal cultures, basonuclin is present in the nuclei of cells in or close to the basal layer but not in the nuclei of cells in more superficial layers. The Ki-67 protein, a nuclear marker for any stage of the multiplication cycle is present in only a subclass of basonuclin-containing cells. In cultured keratinocytes, the disappearance of basonuclin mRNA is associated with loss of colony-forming ability and the appearance of mRNA for involucrin, a protein characteristic of terminal differentiation. In hair follicles, the largest reservoir of basonuclin-containing cells is the outer root sheath, which contains precursors of differentiated cells of the hair shaft and of the epidermis. Basonuclin is not a cell cycle marker but is likely instead to be a regulatory molecular whose presence in the keratinocyte is linked to the maintenance of proliferative capacity and prevention of terminal differentiation.