Altered ribonucleotide reductase activity in mammalian tissue culture cells resistant to hydroxyurea

Four Chinese hamster ovary cell lines and one mouse L cell line have been isolated which are resistant to the cytotoxic effects of hydroxyurea and guanazole. These five cell lines contain an altered ribonucleotide reductase activity as judged by a decreased sensitivity to the inhibitory action of both drugs. This is strong evidence that ribonucleotide reductase is one of the lethal sites of action for these two antitumour agents. The results are also consistent with the view that mammalian cell variants can arise from structural gene mutations.     

Genetic characterization of hydroxyurea-resistance in Chinese hamster ovary cells

Hydroxyurea is an excellent selective agent for obtaining drug-resistant mutants. At a frequency of approximately 1 X 10(-5) it was possible to select, in a single step, colonies that exhibited significant resistance to the cytotoxic effects of the drug. These hydroxyurea-resistant cell lines maintained their resistant phenotype after extensive cultivation in the absence of the drug. Reconstruction experiments indicated that the expression of hydroxyurea-resistance and the frequency of drug-resistant colonies was independent of cell densities up to 5 X 10(5) cells per 100-mm selection plate. Luria-Delbrück fluctuation analyses indicated that the appearance of hydroxyurea-resistant cells in wild type populations occurred spontaneously and at a rate of 4.8 X 10(-6) per cell per generation in the presence of 0.33 mM drug. Studies with the mutagen, ethyl methane sulfonate indicated that it was capable of increasing the frequency of hydroxyurea-resistant cells by a factor of approximately 10. Also, cell-cell hybridization experiments showed that hydroxyurea-resistance behaves as a dominant or codominant trait and that hydroxyurea-resistance was a useful new genetic marker for selection of somatic cell hybrids. Furthermore, similar to many other drug-resistant cell lines hydroxyurea-resistant cells were found to exhibit an altered sensitivity to a number of non-selective agents (guanazole, N-carbamoyloxyurea, formamidoxime, and hydroxyurethane). Except for guanazole these compounds are structurally very similar to hydroxyurea and may be expected to have similar modes of action. The results presented in this paper support the view that hydroxyurea-resistance is expressed as a normal genetic trait and is a useful genetic marker for somatic cell genetic studies.     

N-Carbamoyloxyurea-resistant Chinese hamster ovary cells with elevated levels of ribonucleotide reductase activity

We describe the isolation and characterization of a Chinese hamster ovary cell line selected for resistance to N-carbamoyloxyurea. Using the mammalian cell permeabilization assay developed in our laboratory, a detailed analysis of the target enzyme, ribonucleotide reductase (EC 1.17.4.1), was carried out. Both drug-resistant and parental wild-type cells required the same optimum conditions for enzyme activity. The Ki values for N-carbamoyloxyurea inhibition of CDP reduction were 2.0 mM for NC^R-30A cells and 2.3 mM for wild-type cells, while the Ki value for ADP reduction was 2.3 mM for both cell lines. Although the Ki values remained essentially unchanged, the V_max values for NC^R-30A cells were 1.01 nmoles dCDP formed/5 × 10⁶ cells/hour and 1.83 nmoles dADP/5 × 10⁶ cells/hour, while those for the wild-type cells were 0.49 nmoles dCDP produced/5 × 10⁶ cells/hour and 1.00 nmoles dADP/5 × 10⁶ cells/hour. This approximate twofold increase in reductase activity at least partially accounts for a 2.6-fold increase in D₁₀ value for cellular resistance to N-carbamoyloxyurea exhibited by NC^R-30A cells. The NC^R-30A cell line was also cross-resistant to the antitumor agents, hydroxyurea and guanazole. No differences in Ki values for inhibition of CDP and ADP reduction by these two drugs were detected and cellular resistance could be entirely accounted for by the elevation in activity of the reductase in the NC^R-30A cell line. The properties of N-carbamoyloxyurea-resistance cells indicate they should be useful for further investigations into the regulation of mammalian enzyme activity.     

New insights into mechanisms of resistance to microtubule inhibitors

Mechanisms to explain tumor cell resistance to drugs that target the microtubule cytoskeleton have relied on the assumption that the drugs act either to suppress microtubule dynamics or to perturb the balance between assembled and nonassembled tubulin. Recently, however, it was found that these drugs also alter the stability of microtubule attachment to centrosomes, and do so at the same concentrations that are needed to inhibit cell division. Based on this new information, a new model is presented that explains resistance resulting from a variety of molecular changes that have been reported in the literature. The improved understanding of drug action and resistance has important implications for chemotherapy with these agents.     

Mycobacterial cell wall: Structure and role in natural resistance to antibiotics

Abstract Mycobacteria show a high degree of intrinsic resistance to most antibiotics and chemotherapeutic agents. The low permeability of the mycobacterial cell wall, with its unusual structure, is now known to be a major factor in this resistance. Thus hydrophilic agents cross the cell wall slowly because the myobacterial porin is inefficient in allowing the permeation of solutes and exists in low concentration. Lipophilic agents are presumably slowed down by the lipid bilayer which is of unusually low fluidity and abnormal thickness. Nevertheless, the cell wall barrier alone cannot produce significant levels of drug resistance, which requires synergistic contribution from a second factor, such as the enzymatic inactivation of drugs.     

Molecular mechanisms of drug resistance and its reversal in cancer

Chemotherapy is the main strategy for the treatment of cancer. However, the main problem limiting the success of chemotherapy is the development of multidrug resistance. The resistance can be intrinsic or acquired. The resistance phenotype is associated with the tumor cells that gain a cross-resistance to a large range of drugs that are structurally and functionally different. Multidrug resistance arises via many unrelated mechanisms, such as overexpression of energy-dependent efflux proteins, decrease in uptake of the agents, increase or alteration in drug targets, modification of cell cycle checkpoints, inactivation of the agents, compartmentalization of the agents, inhibition of apoptosis and aberrant bioactive sphingolipid metabolism. Exact elucidation of resistance mechanisms and molecular and biochemical approaches to overcome multidrug resistance have been a major goal in cancer research. This review comprises the mechanisms guiding multidrug resistance in cancer chemotherapy and also touches on approaches for reversing the resistance.     

Mechanisms of resistance to alkylating agents

Alkylating agents are the most widely used anticancer drugs whose main target is the DNA, although how exactly the DNA lesions cause cell death is still not clear. The emergence of resistance to this class of drugs as well as to other antitumor agents is one of the major causes of failure of cancer treatment. This paper reviews some of the best characterized mechanisms of resistance to alkylating agents. Pre- and post-target mechanisms are recognized, the former able to limit the formation of lethal DNA adducts, and the latter enabling the cell to repair or tolerate the damage. The role in the pre-target mechanisms of reduced drug accumulation and the increased detoxification or activation systems (such as DT-diaphorase, metallothionein, GST/GSH system, etc...) are discussed. In the post-target mechanisms the different DNA repair pathways, tolerance to alkylation damage and the ‘downstream’ effects (cell cycle arrest and/or apoptosis) are examined. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microtubule assembly dynamics: an attractive target for anticancer drugs

Microtubules, composed of alphabeta tubulin dimers, are dynamic polymers of eukaryotic cells. They play important roles in various cellular functions including mitosis. Microtubules exhibit differential dynamic behaviors during different phases of the cell cycle. Inhibition of the microtubule assembly dynamics causes cell cycle arrest leading to apoptosis; thus, qualifying them as important drug targets for treating several diseases including cancer, neuronal, fungal, and parasitic diseases. Although several microtubule-targeted drugs are successfully being used in cancer chemotherapy, the development of resistance against these drugs and their inherent toxicities warrant the development of new agents with improved efficacy. Several antimicrotubule agents are currently being evaluated for their possible uses in cancer chemotherapy. Benomyl, griseofulvin, and sulfonamides have been used as antifungal and antibacterial drugs. Recent reports have shown that these drugs have potent antitumor potential. These agents are shown to inhibit proliferation of different types of tumor cells and induce apoptosis by targeting microtubule assembly dynamics. However, unlike vincas and taxanes, which inhibit cancer cell proliferation in nanomolar concentration range, these agents act in micromolar range and are considered to have limited toxicities. Here, we suggest that these drugs may have a significant use in cancer chemotherapy when used in combination with other anticancer drugs.     

质子泵抑制剂与肿瘤耐药研究

恶性肿瘤对抗癌药物的耐药性是肿瘤患者治疗失败的主要原因。肿瘤细胞外微环境的高度酸化是肿瘤细胞对化疗药物产生耐药的机制之一。改变肿瘤细胞内外的pH梯度是逆转耐药的一种有效方法。作为抗酸剂治疗胃病的质子泵抑制剂能够通过抑制质子泵的功能,改变pH梯度而阻断肿瘤微环境的酸化,达到提高肿瘤对化疗药物敏感性的目的。     

Hierarchy of in vitro sensitivity and resistance of tumor cells to cytotoxic effector cells,cytokines, drugs and toxins

Summary Drug resistance of tumor cells has led to the development of other therapeutic modalities including biological response modifiers, lymphokine-activated killer cells (LAK), and cytokines alone and in combination. The premise of these alternative modalities is that drug resistance can be overcome by other cytotoxic agents or cytotoxic effector cells. However, the relationship between tumor cell sensitivity to these different agents and the cytotoxicity caused by drugs is not known or well understood. Thus, understanding the relationship between these different systems of tumor cell cytotoxicity is essential for optimal therapeutic intervention. To this end, we compared the tumor cell cytotoxicity mediated by recombinant tumor necrosis factor (rTNF), cytotoxic effector cells (natural killer cells, monocytes, LAK cells), chemotherapeutic drugs, and microbial toxins. Human tumor cell lines sensitive and resistant to rTNF or drugs were used to evaluate the effectiveness of the other cytotoxic modalities. Sensitivity was considered as tumor cell cytotoxicity above 15% while resistance refers to that below 10%. Cell lines tested consisted of several histological types such as brain, lung, colon and ovarian tumors. In our experiments, cell lines made resistant to rTNF by coculture were also relatively resistant to unactivated monocytes and their supernatants. These lines were sensitive to all other methods tested including activated monocytes, natural killer and LAK cells, drugs, and toxins. The tumor lines naturally resistant to rTNF were found to have various degrees of sensitivity and resistance to these other systems. Upon the analysis of our data, a pattern emerged that suggested a hierarchy of sensitivity and resistance of the tumor cells to the cytotoxic mechanisms explored. From a majority of cell lines resistant to rTNF to a minority of lines resistant to LAK, we found an interesting gradation of sensitivity and/or resistance to the other cytotoxic modalities employed. The hypothesis of an underlying common mechanism of action within these systems is discussed.Supported in part by grant CA43 121 from the Department of Health and Human services, NIH, and NRSA clinical and fundamental immunology training grant A107 126, NIH (J. S.), and in part by a grant from the Concern Foundation, Los Angeles and a gift from the Boiron Foundation     

Drug discovery targeting cell division proteins,microtubules and FtsZ

Eukaryotic cell division or cytokinesis has been a major target for anticancer drug discovery. After the huge success of paclitaxel and docetaxel, microtubule-stabilizing agents (MSAs) appear to have gained a premier status in the discovery of next-generation anticancer agents. However, the drug resistance caused by MDR, point mutations, and overexpression of tubulin subtypes, etc., is a serious issue associated with these agents. Accordingly, the discovery and development of new-generation MSAs that can obviate various drug resistances has a significant meaning. In sharp contrast, prokaryotic cell division has been largely unexploited for the discovery and development of antibacterial drugs. However, recent studies on the mechanism of bacterial cytokinesis revealed that the most abundant and highly conserved cell division protein, FtsZ, would be an excellent new target for the drug discovery of next-generation antibacterial agents that can circumvent drug-resistances to the commonly used drugs for tuberculosis, MRSA and other infections. This review describes an account of our research on these two fronts in drug discovery, targeting eukaryotic as well as prokaryotic cell division.     

LIMK2 Mediates Resistance to Chemotherapeutic Drugs in Neuroblastoma Cells through Regulation of Drug-Induced Cell Cycle Arrest

Drug resistance is a major obstacle for the successful treatment of many malignancies, including neuroblastoma, the most common extracranial solid tumor in childhood. Therefore, current attempts to improve the survival of neuroblastoma patients, as well as those with other cancers, largely depend on strategies to counter cancer cell drug resistance; hence, it is critical to understand the molecular mechanisms that mediate resistance to chemotherapeutics. The levels of LIM-kinase 2 (LIMK2) are increased in neuroblastoma cells selected for their resistance to microtubule-targeted drugs, suggesting that LIMK2 might be a possible target to overcome drug resistance. Here, we report that depletion of LIMK2 sensitizes SHEP neuroblastoma cells to several microtubule-targeted drugs, and that this increased sensitivity correlates with enhanced cell cycle arrest and apoptosis. Furthermore, we show that LIMK2 modulates microtubule acetylation and the levels of tubulin Polymerization Promoting Protein 1 (TPPP1), suggesting that LIMK2 may participate in the mitotic block induced by microtubule-targeted drugs through regulation of the microtubule network. Moreover, LIMK2-depleted cells also show an increased sensitivity to certain DNA-damage agents, suggesting that LIMK2 might act as a general pro-survival factor. Our results highlight the exciting possibility of combining specific LIMK2 inhibitors with anticancer drugs in the treatment of multi-drug resistant cancers.     

Methylation of death-associated protein kinase is associated with cetuximab and erlotinib resistance

Anti-EGFR therapy is among the most promising molecular targeted therapies against cancer developed in the past decade. However, drug resistance eventually arises in most, if not all, treated patients. Emerging evidence has linked epigenetic changes, such as DNA methylation at CpG islands, to the development of resistance to multiple anticancer drugs. In addition, genes that are differentially methylated have increasingly been appreciated as a source of clinically relevant biomarker candidates. To identify genes that are specifically methylated during the evolution of resistance to anti-EGFR therapeutic agents, we performed a methylation-specific array containing a panel of 56 genes that are commonly known to be regulated through promoter methylation in two parental non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cell lines and their resistant derivatives to either erlotinib or cetuximab. We found that death-associated protein kinase (DAPK) was hypermethylated in drug-resistant derivatives generated from both parental cell lines. Restoration of DAPK into the resistant NSCLC cells by stable transfection re-sensitized the cells to both erlotinib and cetuximab. Conversely, siRNA-mediated knockdown of DAPK induced resistance in the parental sensitive cells. These results demonstrate that DAPK plays important roles in both cetuximab and erlotinib resistance, and that gene silencing through promoter methylation is one of the key mechanisms of developed resistance to anti-EGFR therapeutic agents. In conclusion, DAPK could be a novel target to overcome resistance to anti-EGFR agents to improve the therapeutic benefit, and further evaluation of DAPK methylation as a potential biomarker of drug response is needed.     

Methylation of death-associated protein kinase is associated with cetuximab and erlotinib resistance

Anti-EGFR therapy is among the most promising molecular targeted therapies against cancer developed in the past decade. However, drug resistance eventually arises in most, if not all, treated patients. Emerging evidence has linked epigenetic changes, such as DNA methylation at CpG islands, to the development of resistance to multiple anticancer drugs. In addition, genes that are differentially methylated have increasingly been appreciated as a source of clinically relevant biomarker candidates. To identify genes that are specifically methylated during the evolution of resistance to anti-EGFR therapeutic agents, we performed a methylation-specific array containing a panel of 56 genes that are commonly known to be regulated through promoter methylation in two parental non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cell lines and their resistant derivatives to either erlotinib or cetuximab. We found that death-associated protein kinase (DAPK) was hypermethylated in drug-resistant derivatives generated from both parental cell lines. Restoration of DAPK into the resistant NSCLC cells by stable transfection re-sensitized the cells to both erlotinib and cetuximab. Conversely, siRNA-mediated knockdown of DAPK induced resistance in the parental sensitive cells. These results demonstrate that DAPK plays important roles in both cetuximab and erlotinib resistance, and that gene silencing through promoter methylation is one of the key mechanisms of developed resistance to anti-EGFR therapeutic agents. In conclusion, DAPK could be a novel target to overcome resistance to anti-EGFR agents to improve the therapeutic benefit, and further evaluation of DAPK methylation as a potential biomarker of drug response is needed.     

The effect of some beta-lactam antibiotics on Escherichia coli studied by flow cytometry

The effects of three beta-lactam antibiotics on Escherichia coli were studied by means of flow cytometry. Since these agents block bacterial cell wall synthesis in such manner as to prevent septal formation without appreciably affecting nucleic acid synthesis, the resulting cell elongation caused by these agents can be assessed by nucleic acid fluorescent staining. It was shown by this technique that the somatic effects of cefazolin, cefamandole and moxalactam were related both to the antibiotic concentration and time of exposure to the drugs and were observable within 30 minutes of the initial exposure of the cultures to these agents. These results demonstrate that fluorescent cytometry can provide accurate assessment of the effects of compounds that inhibit cell wall formation. This technology could be a useful tool for comparing antibiotic somatic effects on bacteria and for rapidly and reliably determining their sensitivity and resistance to these agents.     

A cross drug resistance of mammalian cells with a high efficiency of "DNA clearing"

The process of active dissociation of noncovalently bound agents from DNA or "DNA clearing" in the living cells was described elsewhere. The vital fluorescent bisbenzimidazole dye Hoechst 33342 (4342), which binds tightly but not covalently to DNA in the minor groove, was used for studying interactions of agents noncovalently binding with DNA. The "DNA clearing" is an energy-dependent process, which is suppressed by topoisomerase-II inhibitors and DNA breaks. It has been shown that the rodent fibroblast cell line AA8HoeR-7 is selected for resistance to H342, and characterized by an enhanced dissociation of the bisbenzimidazole dye-DNA complex. This cell line obtained cross-resistance to other DNA damaging drugs: mitomycin C, etoposide and ethidium bromide. That proves that AA8HoeR-7 is cell line with a new mechanism of multidrug resistance.     

The extended-MDR phenotype

Cellular models have made a significant contribution to our understanding of the molecular mechanisms of resistance to chemotherapeutic drugs. However the vast majority of these models involve cell sublines with high levels of resistance generated by continuous exposure to high drug doses, and although the majority express a multidrug resistance (MDR) phenotype, they fall short of the broader drug cross resistance that is characteristic of cancers which no longer respond to treatment. Several studies have reported cell sublines which not only have the MDR phenotype and are resistant to ‘natural product’ lipophilic drugs, but they are also resistant to alkylating agents and antimetabolites. A common feature of these sublines is they were generated by treatment with low, clinically relevant levels of drug given intermittently. The term extended-MDR has been used to describe this type of broad drug cross resistance. Here we review those factors that promote the development of extended-MDR, the characteristics of extended-MDR sublines and the possible resistance mechanisms involved. This revised version was published online in August 2006 with corrections to the Cover Date.     

Overcoming acquired resistance to kinase inhibition: The cases of EGFR,ALK and BRAF

In the past decade, several kinase inhibitors have been approved based on their clinical benefit for cancer patients. Unfortunately, in many cases, patients develop resistance to these agents via secondary mutations and alternative mechanisms. This review will focus on the cases of acquired resistance to EGFR and ALK inhibitors for non-small cell lung cancer patients and BRAF inhibitors for melanoma patients. I will overview the main causes of acquired resistance, and explore the chemical scaffolds as well as combination of drugs, used to tackle these major causes of resistance.     

Induction of apoptosis by chemotherapeutic drugs: the role of FADD in activation of caspase-8 and synergy with death receptor ligands in ovarian carcinoma cells

Although ovarian tumours initially respond to chemotherapy, they gradually acquire drug resistance. The aims of this study were to identify how chemotherapeutic drugs with diverse cellular targets activate apoptotic pathways and to investigate the mechanism by which exposure to a combination of drugs plus death receptor ligands can increase tumour cell kill. The results show that drugs with distinct cellular targets differentially up-regulate TRAIL and TNF as well CD95L, but do not require interaction of these ligands with their receptor partners to induce cell death. Factors that were critical in drug-induced apoptosis were activation of caspases, with caspase-8 being activated by diverse drugs in a FADD-independent manner. Certain drugs also demonstrated some dependence on FADD in the induction of cell death. Caspase-9 was activated more selectively by chemotherapeutic agents. Combining ligation of death receptors with exposure to drugs increased tumour cell kill in both drug resistant cell lines and primary ovarian carcinoma cells, even though these cells were not sensitive to death receptor ligation alone. CD95L was more consistent at combining with drugs than TRAIL or TNF. Investigation of the mechanism by which a combination of drugs plus CD95 ligation can increase cell death showed that caspase-8 was activated in cells exposed to a combination of cisplatin and anti-CD95, but not in cells exposed to either agent alone.