Cellular and molecular determinants of cisplatin resistance

Cisplatin and carboplatin are among the most active and widely used cytotoxic anticancer drugs. However, the acquisition or presence of resistance significantly undermines the curative potential of these drugs against many malignancies. Multiple potential mechanisms of resistance have been identified at the cellular and molecular levels. Alterations in cellular pharmacology, including decreased drug accumulation, increased cellular thiol levels and increased repair of platinum-DNA damage, have been observed in numerous model systems. More recently, it has become apparent that an enhanced capacity to tolerate cisplatin-induced damage may also contribute to resistance. Alterations in proteins that recognise cisplatin-DNA damage (mismatch repair and high-mobility group (HMG) family proteins) and in pathways that determine sensitivity to apoptosis may contribute to damage tolerance. It remains to be determined whether any of these mechanisms contribute significantly to resistance in the clinical setting. Ongoing biochemical modulation and translational correlative trials should clarify which specific mechanisms are most relevant to clinical cisplatin resistance. Such investigations have the potential to improve the ability to predict likelihood of response and should identify potential targets for pharmacological or molecular intervention.     

Reduction in post-intubation respiratory resistance by isoflurane and albuterol

PURPOSE: This study examined the bronchodilating effects of 0.6 MAC and 1.1 MAC isoflurane (ISF) on respiratory system resistance (Rrs) following tracheal intubation and determined whether albuterol supplements that effect. METHODS: Sixty-seven adult patients were anaesthetized with 2 micrograms.kg-1 fentanyl and 5 mg.kg-1 thiopentone and their tracheas intubated following administration of 1 mg.kg-1 succinylcholine. Respiratory system resistance was measured following intubation and the patients then randomized to receive either 1.1 MAC ISF in oxygen or 0.6 MAC ISF in 50% nitrous oxide and oxygen. Ten minutes later, Rrs was again measured. Patients were then further randomized to receive albuterol or a placebo using incremental doses of 2, 5, and 10 puffs (albuterol puff = 90 micrograms) delivered via a metered dose inhaler at ten minute intervals. RESULTS: Isoflurane at 1.1 MAC decreased post-intubation Rrs by 23 +/- 5% (mean +/- sem) whereas the decrease was only 7 +/- 5% for 0.6 MAC ISF (P < 0.01). Two puffs of albuterol resulted in a further decrease of 12 +/- 3% (mean +/- sem) in Rrs compared with a 2 +/- 4% decrease in the placebo groups (P < 0.05). Additional puffs of albuterol resulted in no further changes in Rrs. CONCLUSION: We conclude that following tracheal intubation the reduction in Rrs produced by ISF is highly concentration dependent. Albuterol results in a small further reduction in Rrs.     

Reduction of anticoccidial drug resistance by massive introduction of drug-sensitive coccidia

Massive introduction of a drug-sensitive attenuated strain of Eimeria tenella in a floor-pen heavily contaminated with a drug-resistant strain produced a marked reduction in the proportion of drug-resistant oocytsts in the litter. This provides a useful adjunct to planned immunization programs since the procedure protects the birds against subsequent challenge through immunity imparted by the introduced strain while restoring the effectiveness of the anticoccidial drug which was previously ineffective because of the predominance of drug-resistant coccidia.     

In vitro circumvention of cisplatin resistance by the novel sterically hindered platinum complex AMD473

BACKGROUND: Oral indomethacin causes villous shortening, microvascular damage, and distortion, which might induce mucosal ischaemia and necrosis. AIMS: In order to determine the early events in indomethacin induced jejunal injury we examined the temporal relations between morphological damage and changes in villous blood flow following indomethacin. METHODS: In anaesthetised rats, mid jejunal villi were exteriorised in a chamber and observed by fluorescence microscopy. Blood flow in surface capillaries was calculated from velocities and diameters. Indomethacin was applied by both luminal and intravenous routes for 90 minutes, after which the animal was perfusion fixed and the villi were processed for histological examination. Control animals received intravenous or luminal bicarbonate (1.25%). RESULTS: Blood flow slowed in individual villi at 20 minutes, and progressed to complete stasis (in another group) by 45 minutes. Histological examination at 20 minutes revealed microvascular distortion, but no villous shortening; crypt depth:villous height ratios were 0.356 (0.02) in test and 0.386 (0.01) in surrounding villi (p > 0.05). At stasis, the villi under study showed epithelial clumping and were shortened: crypt depth:villous height ratios were 0.92 (0.2) in test and 0.42 (0.06) in surrounding villi (p < 0.02). Vehicle alone had no effect on either blood flow or histology. CONCLUSIONS: Focal slowing of villous blood flow and microvascular distortion precede villus shortening and epithelial disruption, and indicate that damage to surface microvasculature is an early event in indomethacin induced mucosal injury in this model.     

Insights into potential cellular mechanisms of cisplatin nephrotoxicity and their clinical application

Cisplatin preferentially accumulates in cells of the S3 segment of the renal proximal tubule and is toxified intracellularly by hydration. The earliest manifestation of toxicity is inhibition of protein synthesis. GSH depletion is another important mechanism causing CP toxicity. Intracellular binding to SH groups leads to GSH depletion, resulting in lipid peroxidation and eventually mitochondrial damage. New measures to prevent GSH depletion and scavenge intracellular free oxygen radicals have been tried in clinical studies. Promising results indicate that cisplatin nephrotoxicity can be further reduced in the future.     

Reversal of radiation-induced cisplatin resistance in murine fibrosarcoma cells by selective modulation of the cyclic GMP-dependent transduction pathway

Cisplatin resistance, induced in murine fibrosarcoma cells (SSK) in vitro or in vivo by low-dose irradiation, can be overcome by activation of the cyclic GMP(cGMP)-dependent transduction pathway. This is mediated either by stimulating cGMP formation with sodium nitroprusside or by replacing cGMP with a selective activator of the cGMP-dependent protein kinase, 8-bromo-cGMP. The cyclic AMP-dependent transduction pathway is not involved in cisplatin resistance. Instead, activation of cAMP sensitises both parental and resistant SSK cells equally to the action of cisplatin. There is a 1.8 to 2.5-fold increase in drug toxicity, depending on the activating agent. Enhancement of cisplatin sensitivity is induced by specific inhibition of cAMP hydrolysis, increase in cAMP formation or by increasing the activation potential to cAMP-dependent protein kinase by specific cAMP analogues. Cells that have lost cisplatin resistance respond to cGMP- or cAMP-elevating agents in the same way as the parental SSK cells. The radiation sensitivity is unchanged in all cell lines, even after activation of cAMP or cGMP. These results suggest that specific DNA repair pathways are altered by radiation but affected only in cisplatin damage repair, which is regulated by cGMP. Although there is ample cooperativity and interaction between the cAMP- and the cGMP-dependent transduction pathways, specific substrate binding by cGMP appears to play an important role in radiation-induced cisplatin resistance.     

The cellular interaction of 5-fluorouracil and cisplatin in a human colon carcinoma cell line

The combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) has been shown to have synergistic cytotoxicity in human tumours, but the biochemical mechanism for this interaction remains unclear. Therefore, the aim of this study was to investigate the interaction of 5-FU and CDDP in a human colon carcinoma cell line, NCI H548. A 24 h exposure to 5-FU resulted in a 5-FU IC50 value of 24.2 +/- 4.5 microM, Dm 22.6 microM; exposure to CDDP for 2 h resulted in a IC50 value of 20.8 +/- 8.0 microM, Dm 21.9 microM. When cells were exposed simultaneously to 5-FU for 24 h and CDDP for the initial 2 h, or when cells were treated with CDDP for 2 h followed by various concentrations of 5-FU for 24 h, no greater than additive cytotoxicity was observed. In contrast, when cells were treated with 5-FU for 24 h prior to CDDP for 2 h, a greater than additive interaction was noted (5-FU IC50 1.2 +/- 0.6 microM, Dm 1.3 microM, CI 0.45). Thymidine 10 microM partially reversed the growth inhibitory effects of the 5-FU/ CDDP combination. Using both immunological and biochemical assays, no notable differences in the total amount of TS enzyme or the fraction of bound TS enzyme could be detected in the absence or presence of CDDP. No notable differences could be detected in intracellular reduced folate pools, FdUMP or FUTP pools, or 5-FU incorporation into RNA or DNA with the addition of CDDP to 5-FU. DNA fragmentation studies revealed that the combination of 5-FU followed by CDDP demonstrated a greater degree of single-stranded DNA fragments in parental (P = 0.024) and newly synthesised DNA (P = 0.025) compared with the administration of CDDP prior to 5-FU or either drug alone. The increase in smaller DNA fragment size was reversed with the addition of thymidine (10 microM). These findings suggest that the interaction of 5-FU and CDDP is associated with a greater degree of fragmentation of both nascent and parental DNA.     

Role of apoptotic response in cellular resistance to cytotoxic agents

To elucidate compositional changes of the pubic symphysis (PS) by aging, elements of pubic symphysis (PSs) removed from 26 cadavers were determined by inductively coupled plasma atomic-emission spectrometry. It was found that the relative contents (RCs) of calcium and phosphorus in women's PSs were about three- and five-fold amounts as compared with those in men's PSs, respectively. In contrast, the RCs of sulfur, magnesium, sodium, and iron in women's PSs were somewhat lower than those in men's PSs. The accumulations of calcium (Ca) and phosphorus (P) in women's PSs occurred mainly beyond the age of 70-yr-old, but did not occur in men's PSs.     

Potentiation of cisplatin cytotoxicity by 9-aminocamptothecin

Camptothecin (CPT) derivatives are presently in ongoing Phase I/II clinical trials. The interactions between 9-aminocamptothecin (9AC) and cisplatin (CDDP) have been studied in the IGROV-1 human ovarian cancer cell line used in the National Cancer Institute Drug Discovery Anticancer Screen. One-h simultaneous treatment with 9AC and CDDP produced synergistic cytotoxicity. Under these conditions, 9AC delayed the reversal of CDDP-induced DNA interstrand cross-links (ISCs) without modifying the maximum ISC frequency at 6 h after drug treatment. CDDP did not affect the amount and the kinetics of reversion of 9AC-induced DNA single-strand breaks. Simultaneous treatment with CDDP and 9AC prolonged the DNA synthesis inhibition produced by each drug alone. Consistently, flow cytometry analyses showed enhanced S-phase arrest in cells treated with the CDDP-9AC combination. The DNA polymerase inhibitor aphidicolin also increased the residual CDDP-induced ISCs. These results suggest that prolonged inhibition of DNA synthesis by CPTs potentiate the cytotoxicity of CDDP by inhibiting the reversal of CDDP-induced DNA damage. Therefore, the combination of CPTs and CDDP appears to be worthwhile in cancer chemotherapy.     

Electric resistance and thermal conductivity of highly porous permeable cellular materials

Translated from Poroshkovaya Metallurgiya, No. 8(308), pp. 87–92, August, 1988.     

Long recovery times improve the detection of cellular resistance in vitro

In vitro cytotoxicity testing is used increasingly during the development of clinical treatment protocols. These tests are influenced by many variables, not all of which have been assessed systematically yet. We analyzed the influence of the recovery time between the end of treatments and measurements on the detection of cellular resistance. The development of resistance to cisplatin and radiation was chosen as a model since the schedule of these treatments is the objective of several ongoing clinical trials. C6 rat glioma, T98G, 86HG-39, A172 human glioma and TE671 human rhabdomyo-sarcoma cells were pretreated with radiation or cisplatin. The cellular resistance was then compared in pretreated and wild-type cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. In all cell lines, apparent drug concentrations killing 50% of the cells were dependent on the recovery time. In A172 cells this concentration was 10.3+/-02.1 microM after 48 h but decreased to 3.56+/-0.44 microM after 120 h recovery time (P < 0.001). After recovery times of more than 168 h, 53% of all pretreated cell lines were resistant to cisplatin or radiation, 25% were unchanged and 22% were more sensitive. However, only half the resistant cells could be identified when the MTT test was done with only 48 h recovery time. The sensitivity of detection increased from 0.46 to 0.83 when the recovery time of the test system was extended from 48 h to 168 h. The specificity was not dependent on the recovery time. Experiments showing resistance after short recovery times are reliable, but lack of resistance can only be shown in experiments with long recovery times. Cisplatin treatment can result in resistance to radiation in glioma cells.     

Mechanisms of resistance of human small cell lung cancer lines selected in VP-16 and cisplatin

BACKGROUND: The combination of VP-16 and cisplatin is one of the most active regimens available for the treatment of small cell lung cancer (SCLC), however, most tumors eventually become resistant to these drugs. METHODS: To investigate the problem of resistance to VP-16 and cisplatin in patients with SCLC, we established two resistant sublines from the drug sensitive human SCLC line, NCI-H209, by in vitro selection in VP-16 and cisplatin. RESULTS: The VP-16-selected cell line, H209/VP, was more than 100-fold resistant to VP-16, and displayed cross-resistance to VM-26 and other topoisomerase II interactive drugs, but not to vinca alkaloids. There was no difference in accumulation of VP-16 in H209/VP compared with its parent cell line. The level of topoisomerase II-alpha was reduced to 8% of that in the parent cell line, and there was an altered form of this enzyme with a molecular weight of 160 kilodaltons (kDa), in addition to the normal 170 kDa protein. The cisplatin-selected cell line, H209/CP, was 11.5-fold resistant to cisplatin, with only a low level of cross-resistance to other platinum compounds including carboplatin, tetraplatin, iproplatin, and lobaplatin. This line was highly cross-resistant to vinca alkaloids, but not to anthracyclines or epipodophyllotoxins. The H209/CP cell line was not resistant to cadium chloride, suggesting that alterations in metallothionein are unlikely to be a cause of resistance. Although glutathione (GSH) levels were increased nearly 2-fold in H209/CP, there was no difference in levels of the GSH-related enzymes glutathione-S-transferase, glutathione peroxidase, and glutathione reductase, compared with the parent line. The H209/CP line had a 1.4-fold elevation of topoisomerase II-alpha. The accumulation of cisplatin was reduced in this cell line, and there were fewer DNA-interstrand cross links formed in the presence of cisplatin in H209/CP, compared with the parent line. Neither H209/VP nor H209/CP expressed MDR1, the gene for P-glycoprotein. The MRP gene was expressed at a slightly higher level in the H209/VP cell line, but there was no significant increase in expression of this gene in the H209/CP cell line. CONCLUSIONS: The resistance of the H209/VP cell line is associated with an alteration of topoisomerase II-alpha, whereas the resistance in the H209/CP line is associated with reduced drug accumulation.     

Emergence of resistance to VP-16/cisplatin chemotherapy: study in a lymphoblast model system

Etoposide (VP-16) and cisplatin are widely used in the treatment of malignancy. It is a common clinical observation that patients may initially respond to this two drug combination but later become resistant to it. Data from the CCL-159 lymphoblast cell line suggests that the emergence of resistance may be by means other than multiple drug resistance gene expression. The data suggest that chemotherapeutic failure may be mediated in part by a relatively inefficient method of drug resistance, the unstable expression of low-level drug resistance by a minority of cells. These results may help explain how patients whose malignancies are initially sensitive to VP-16/cisplatin later develop drug resistance.     

Simulation of recrystallization by cellular automata

A cellular automata (CA) model for recrystallization is introduced. Its behviour is investigated under the influence of different model parameters and algorithms that simulate the kinetics of nucleation and growth of recrystallizing grains. The rate of released energy is calculated by the CA-model so that comparisons with calorimetric measurements are possible. The CA-model givesthe same results as the theory of Johnson, Mehl, Avrami and Kolmogorov (JMAK) for the same model assumptions. Systematic deviations of experimental data from the JMAK-theory are simulated by special model assumptions of the CA. This leads to good agreement with experimental data. It is shown that the CA-model is easy to handle, very simple in the basic algorithm and extremely flexible for introducing special model assumptions.     

Absence of direct relationship between intraperitoneal cellular influx and resistance to experimental peritonitis

In 1995, 23 post-RN (RN-to-BSN) students successfully completed the first clinical nursing course offered by distance methods at Memorial University of Newfoundland. Distance delivery improved access to the course for RNs dealing with conflicting work and family demands. Students rated the course favorably overall and found they had learned and applied new knowledge and skills. Community health nurses, who acted as field guides, and their supervisors were satisfied with their roles and responsibilities. However, one need identified was for detailed information about the course, and another was for more frequent communication with the course instructors during the term. Evaluation found students were able to meet their course objectives and perform as well as students who had completed a similar on-campus course.     

高铁硫化锌精矿的真空铁还原

开展了真空下铁还原高铁硫化锌精矿的热力学分析和试验研究.在整个还原过程中锌以单质形式冷凝回收,直收率达95%左右,矿物中的硫主要以FeS的形式固定.通过试验研究确定了真空下铁还原高铁硫化锌精矿较好的工艺条件.     

Cu还原MoO_4~(2-)产物特性研究

对在酸性条件下Cu还原Mo O42-的行为及还原产物的特性进行了研究。考察了还原体p H值、还原时间、反应温度等因素对钼还原率的影响。结果表明,还原反应要在酸度比较高的条件下才能进行,提高酸度有利于钼的还原;还原速率随温度的升高而增大,符合经典的Arrhenius温度对反应速率的影响规则。同时,采用X-衍射、X荧光分析、拉曼光谱以及扫描电镜对还原产物的特性进行了表征,确定了还原产物的组成与形态,即还原产物中主要的元素有Na、Mo、O,比例是0.2∶4.96∶1,产物组成可定性为Na0.91O19.2H7Mo5.33。产物晶系为六棱型结构,产物中Mo呈现出+5.72价的混合价态。碱分解机理分析得,Cu还原Mo O42-的产物易被氧化,生成Mo O42-。     

Reduced expression of the ICE-related protease CPP32 is associated with radiation-induced cisplatin resistance in HeLa cells

Low-dose fractionated gamma-irradiation (three cycles of 5 x 2 Gy) induced cisplatin resistance in HeLa cells. The drug resistance was modest (Rf of about 2) and stable, similar to that found previously in murine cells after irradiation. In the drug-resistant HeLa-C3 cells, flow cytometric analysis revealed a decreased number of apoptotic cells compared with the parental cells. Drug resistance was associated with considerably enhanced expression of the p53 suppressor protein in HeLa-C3 cells after cisplatin exposure but seemed not to be regulated by the bcl-2-dependent pathway. Cisplatin resistance correlated with reduced expression of ICE-related proteases (interleukin-1beta-converting enzyme). Basal levels of the 45-kDa precursor ICE protein were reduced in HeLa-C3 cells, while those of the mature 60-kDa heterotetramer were similar. The CPP32 protease, a member of the ICE family with structural homology but different substrate specificity, was expressed at a lowered level. After drug exposure, there was a slight increase of CPP32 in HeLa-C3 cells, equivalent to about 45% of the level attained in the parental cells. This is in contrast to the CPP32 levels measured after irradiation, which were similar in sensitive and in resistant cells. As the radiosensitivity is unchanged in both cell lines, these results suggest that cisplatin resistance in HeLa-C3 cells is associated with alterations of a CPP32-linked apoptotic pathway, which is affected by the damage caused by cisplatin but not by irradiation. Whether these changes are dependent on the observed p53 modifications is now being studied in resistant clones.