Phase II study of high-sensitivity genotyping of KRAS, NRAS, BRAF and PIK3CA to ultra-select metastatic colorectal cancer patients for panitumumab plus FOLFIRI: the ULTRA trial.

BACKGROUND: Several studies show the importance of accurately quantifying not only KRAS and other low-abundant mutations because benefits of anti-EGFR therapies may depend on certain sensitivity thresholds. We assessed whether ultra-selection of patients using a high-sensitive digital PCR (dPCR) to determine KRAS, NRAS, BRAF and PIK3CA status can improve clinical outcomes of panitumumab plus FOLFIRI. PATIENTS AND METHODS: This was a single-arm phase II trial that analysed 38 KRAS, NRAS, BRAF and PIK3CA hotspots in tumour tissues of irinotecan-resistant metastatic colorectal cancer patients who received panitumumab plus FOLFIRI until disease progression or early withdrawal. Mutation profiles were identified by nanofluidic dPCR and correlated with clinical outcomes (ORR, overall response rate; PFS, progression-free survival; OS, overall survival) using cut-offs from 0% to 5%. A quantitative PCR (qPCR) analysis was also performed. RESULTS: Seventy-two evaluable patients were enrolled. RAS (KRAS/NRAS) mutations were detected in 23 (32%) patients and RAS/BRAF mutations in 25 (35%) by dPCR, while they were detected in 7 (10%) and 11 (15%) patients, respectively, by qPCR. PIK3CA mutations were not considered in the analyses as they were only detected in 2 (3%) patients by dPCR and in 1 (1%) patient by qPCR. The use of different dPCR cut-offs for RAS (KRAS/NRAS) and RAS/BRAF analyses translated into differential clinical outcomes. The highest ORR, PFS and OS in wild-type patients with their lowest values in patients with mutations were achieved with a 5% cut-off. We observed similar outcomes in RAS/BRAF wild-type and mutant patients defined by qPCR. CONCLUSIONS: High-sensitive dPCR accurately identified patients with KRAS, NRAS, BRAF and PIK3CA mutations. The optimal RAS/BRAF mutational cut-off for outcome prediction is 5%, which explains that the predictive performance of qPCR was not improved by dPCR. The biological and clinical implications of low-frequent mutated alleles warrant further investigations. CLINICALTRIALS.GOV NUMBER: NCT01704703. EUDRACT NUMBER: 2012-001955-38.

Towards the Limits of Existence of Nuclear Structure: Observation and First Spectroscopy of the Isotope

The most remote isotope from the proton dripline (by 4 atomic mass units) has been observed: ^{31}K. It is unbound with respect to three-proton (3p) emission, and its decays have been detected in flight by measuring the trajectories of all decay products using microstrip detectors. The 3p emission processes have been studied by the means of angular correlations of ^{28}S+3p and the respective decay vertices. The energies of the previously unknown ground and excited states of ^{31}K have been determined. This provides its 3p separation energy value S_{3p} of -4.6(2) MeV. Upper half-life limits of 10 ps of the observed ^{31}K states have been derived from distributions of the measured decay vertices.

Scattering of the Halo Nucleus

Angular distributions of the elastic, inelastic, and breakup cross sections of the halo nucleus ^{11}Be on ^{197}Au were measured at energies below (E_{lab}=31.9 MeV) and around (39.6 MeV) the Coulomb barrier. These three channels were unambiguously separated for the first time for reactions of ^{11}Be on a high-Z target at low energies. The experiment was performed at TRIUMF (Vancouver, Canada). The differential cross sections were compared with three different calculations: semiclassical, inert-core continuum-coupled-channels and continuum-coupled-channels ones with including core deformation. These results show conclusively that the elastic and inelastic differential cross sections can only be accounted for if core-excited admixtures are taken into account. The cross sections for these channels strongly depend on the B(E1) distribution in ^{11}Be, and the reaction mechanism is sensitive to the entanglement of core and halo degrees of freedom in ^{11}Be.

Observation and Spectroscopy of New Proton-Unbound Isotopes ³°Ar and ²9Cl: An Interplay of Prompt Two-Proton and Sequential Decay.

Previously unknown isotopes (30)Ar and (29)Cl have been identified by measurement of the trajectories of their in-flight decay products (28)S+p+p and (28)S+p, respectively. The analysis of angular correlations of the fragments provided information on decay energies and the structure of the parent states. The ground states of (30)Ar and (29)Cl were found at 2.25(-0.10)(+0.15) and 1.8±0.1 MeV above the two- and one-proton thresholds, respectively. The lowest states in (30)Ar and (29)Cl point to a violation of isobaric symmetry in the structure of these unbound nuclei. The two-proton decay has been identified in a transition region between simultaneous two-proton and sequential proton emissions from the (30)Ar ground state, which is characterized by an interplay of three-body and two-body decay mechanisms. The first hint of a fine structure of the two-proton decay of (30)Ar*(2(+)) has been obtained by detecting two decay branches into the ground and first-excited states of the (28)S fragment.

Pharmacogenomics in colorectal cancer: a genome-wide association study to predict toxicity after 5-fluorouracil or FOLFOX administration.

The development of genotyping technologies has allowed for wider screening for inherited causes of variable outcomes following drug administration. We have performed a genome-wide association study (GWAS) on 221 colorectal cancer (CRC) patients that had been treated with 5-fluorouracil (5-FU), either alone or in combination with oxaliplatin (FOLFOX). A validation set of 791 patients was also studied. Seven SNPs (rs16857540, rs2465403, rs10876844, rs10784749, rs17626122, rs7325568 and rs4243761) showed evidence of association (pooled P-values 0.020, 9.426E-03, 0.010, 0.017, 0.042, 2.302E-04, 2.803E-03) with adverse drug reactions (ADRs). This is the first study to explore the genetic basis of inter-individual variation in toxicity responses to the administration of 5-FU or FOLFOX in CRC patients on a genome-wide scale.

A novel risk prediction tool for contrast-induced nephropathy in patients with chronic kidney disease who underwent diagnostic coronary angiography.

OBJECTIVE: The incidence of contrast-induced nephropathy (CIN) is higher than 20% in patients with chronic kidney disease. In this study, we sought to define the predictors of CIN and develop a risk prediction tool in patients with chronic kidney disease. PATIENTS AND METHODS: Patients aged 18 years and older who underwent invasive coronary angiography with an iodine-based contrast media between March 2014 and June 2017 were retrospectively analyzed. Independent predictors for CIN development were identified and a new risk prediction tool was created that included these predictors. RESULTS: In total, 283 patients included in the study were divided into those who developed CIN (n=39, 13.8%) and those who did not (n=244, 86.2%). Male gender (OR: 4.874, 95% CI: 2.044-11.621), LVEF (OR: 0.965, 95% CI: 0.936-0.995), diabetes mellitus (OR: 1.711, 95% CI: 1.094-2.677), and e-GFR (OR: 0.880, 95% CI: 0.845-0.917), were identified as independent predictors for the development of CIN in the multivariate analysis. A new scoring system has been designed that can score a minimum of 0 and a maximum of 8 points. Patients with a new scoring system score of ≥4 were at approximately 40 times higher risk of developing CIN than others (OR: 39.9, 95% CI: 5.4-295.3). The area under the curve value of CIN's new scoring system was 0.873 (95% CI, 0.821-0.925). CONCLUSIONS: We found that four easily accessible and routinely collected variables, including sex, diabetes status, e-GFR, and LVEF, were independently associated with the development of CIN. We believe that using this risk prediction tool in routine clinical practice may guide physicians to use preventive medications and techniques in high-risk patients for CIN.

Clinical utility of comprehensive circulating tumor DNA genotyping compared with standard of care tissue testing in patients with newly diagnosed metastatic colorectal cancer.

BACKGROUND: Comprehensive biomarker testing is essential in selecting optimal treatment for patients with metastatic colorectal cancer (mCRC); however, incomplete genotyping is widespread, with most patients not receiving testing for all guideline-recommended biomarkers, in part due to reliance on burdensome sequential tissue-based single-biomarker tests with long waiting times or availability of only archival tissue samples. We aimed to demonstrate that liquid biopsy, associated with rapid turnaround time (TAT) and lower patient burden, effectively identifies guideline-recommended biomarkers in mCRC relative to standard of care (SOC) tissue testing. PATIENTS AND METHODS: Prospectively enrolled patients with previously untreated mCRC undergoing physician discretion SOC tissue genotyping submitted pretreatment blood samples for comprehensive circulating tumor DNA (ctDNA) analysis with Guardant360 and targeted RAS and BRAF analysis with OncoBEAM. RESULTS: Among 155 patients, physician discretion SOC tissue genotyping identified a guideline-recommended biomarker in 82 patients, versus 88 identified with comprehensive ctDNA (52.9% versus 56.8%, noninferiority demonstrated down to α = 0.005) and 69 identified with targeted PCR ctDNA analysis (52.9% versus 44.5%, noninferiority rejected at α = 0.05). Utilizing ctDNA in addition to tissue increased patient identification for a guideline-recommended biomarker by 19.5% by rescuing those without tissue results either due to tissue insufficiency, test failure, or false negatives. ctDNA median TAT was significantly faster than tissue testing when the complete process from sample acquisition to results was considered (median 10 versus 27 days, P < 0.0001), resulting in accelerated biomarker discovery, with 52.0% biomarker-positive patients identified by ctDNA versus 10.2% by SOC tissue 10 days after sample collection (P < 0.0001). CONCLUSIONS: Comprehensive ctDNA genotyping accurately identifies guideline-recommended biomarkers in patients with mCRC at a rate at least as high as SOC tissue genotyping, in a much shorter time. Based on these findings, the addition of ctDNA genotyping to clinical practice has significant potential to improve the care of patients with mCRC.

Comparative analysis of Met-enkephalin, galanin and GABA immunoreactivity in the developing trout preoptic-hypophyseal system.

We studied the organization of Met-enkephalin-containing cells and fibers in the developing preoptic-hypophyseal system of the brown trout (Salmo trutta fario) by immunohistochemistry and determined the relationship of these cells and fibers to the galaninergic and GABAergic systems. Met-enkephalin immunoreactivity was observed in cells in the preoptic area, the hypothalamus and the pituitary of late larvae. In the hypophysis, a few Met-enkephalin-containing cells were present in all divisions of the adenohypophysis, and some immunoreactive fibers were present in the interdigitations of the neural lobe with the proximal pars distalis. Concurrently, GABAergic fibers innervated the anterior and posterior neural lobe. Galanin cells coexisted with Met-enkephalin cells in neuronal groups of the preoptic-hypophyseal system. Galaninergic and GABAergic fibers innervated the preoptic and hypothalamic areas, but GABAergic fibers containing galanin were not observed. These results indicate that Met-enkephalin, galanin and GABA may modulate neuroendocrine activities in the preoptic area, hypothalamus and pituitary during the transition from larval to juvenile period. To better know how the development of the trout preoptic-hypophyseal system takes place, we studied the patterns of cell proliferation and expression of Pax6, a conserved transcription factor involved in the hypophysis development. Pax6 expressing cells and proliferating cells were present in the Rathke's pouch, the hypothalamus and the hypophysis of early larvae. In late larvae, Pax6 expression was no longer observed in these areas, and the density of proliferating cells largely decreased throughout development, although they remained in the hypophysis of late larvae and juveniles, suggesting that Pax6 might play an important role in the early regionalization of the pituitary in the trout.

Effect of Decorin and Bevacizumab on oxygen-induced retinopathy in rat models: A comparative study.

Purpose: The aims of this study were to evaluate the effects of decorin (DCN) in rat oxygen-induced retinopathy (OIR) model and to compare the results with those of bevacizumab. Methods: Twenty-eight newborn Sprague-Dawley rats were randomly divided into four groups. Group I (control): normoxia plus intraperitoneal (ip) normal saline (NS), Group II (sham): OIR plus ip NS, Group III (DCN): OIR plus ip 0.1 mg/kg DCN, and Group IV (bevacizumab): OIR plus ip 2.5 mg/kg bevacizumab. The OIR model was induced by cycling the oxygen concentration between 50% and 10% every 24 h for 14 days following their birth. In all groups, injections were administered on postnatal day (PD) 15. All animals were sacrificed and their right eyes were enucleated on PD 18. The nuclei of neovascular endothelial cells on the vitreal side of the inner limiting membrane were counted, and vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF)-α immunoreactivity were detected in histopathological and immunohistochemical examinations. One-way analysis of variance and post hoc Tukey tests were used for statistical analyses of the data. Results: In Groups II, III, and IV, the mean neovascular cell nuclei counts were 13.14 ± 1.34, 6.57 ± 1.51, and 6.71 ± 1.49, respectively. The mean neovascular cell nuclei count was significantly reduced in treatment groups compared with sham group (P < 0.001). In immunohistochemical staining, the immunoreactivity of VEGF was 0.07 ± 0.02, 0.97 ± 0.21, 0.37 ± 0.12, and 0.23 ± 0.17, respectively. Likewise, immunoreactivity of TNF-α was 0.02 ± 0.02, 1.11 ± 0.36, 0.37 ± 0.13, and 0.62 ± 0.21, respectively. VEGF and TNF-α immunoreactivity increased markedly in the sham group compared with those in the control group (P < 0.001). VEGF and TNF-α immunoreactivity of treatment groups decreased significantly compared to sham group (P < 0.001). Conclusion: The beneficial effects obtained by DCN administration in OIR model were comparable to the effects of bevacizumab.

Effects of compressed seasonally changing day-length cycles on spawning performance, production of viable eggs and levels of vitellogenin in plasma in female yellowtail snapper Lutjanus argentiventris.

Reproduction in yellowtail snapper Lutjanus argentiventris took place after compressing the seasonally changing day length into a 3 month period applied during two consecutive winters, with the longest and shortest days in December and February, respectively. During the first winter, there was no clear peak of days of spawning and the production of viable eggs was similar from the longest and throughout the decreasing day lengths until reproduction ceased. The level of plasma vitellogenin rose abruptly to a maximum concentration during the increasing day length and then decreased dramatically before the longest day length. During the second winter, a clear peak in the number of days of spawning and the highest production of viable eggs occurred around the longest day length. These results showed that it is feasible to synchronize day length between winter-induced and natural summer and autumn reproduction to produce eggs and larvae during the year.

Antimicrobial activity of the extracts and physalin D from Physalis alkekengi and evaluation of antioxidant potential of physalin D.

Physalis alkekengi L. (Solanaceae) is a popular plant in traditional European and Chinese folk medicine, and it has been reported to have many ethnopharmacological properties including antifungal, anti-cough, anti-inflammatory, analgesic, and febricide activities. Some active components from Physalis species have been investigated. However, no antimicrobial activity studies on extracts and physalins of P. alkekengi have been carried out. In this study, we attempted to identify the possible antimicrobial activities of the methanol extract from aerial parts of P. alkekengi and the dichloromethane extract from calyces of the plant. The extracts were tested against five Gram-positive and five Gram-negative bacteria and five Candida species by using disk diffusion and broth microdilution methods. The extracts were fractionated to isolate physalins using chromatographic techniques, and physalin D was isolated from the extracts. The structure of the compound was elucidated on the basis of (1)H-NMR spectroscopic study, and confirmed by comparison with a reference sample and literature data. Results indicated that all the extracts and physalin D were characterized by antibacterial action, especially against Gram-positive bacteria, with MIC values between 32 and 128 microg/mL. The methanol extract had moderate activity against fungi at MICs ranging from 128 to 512 microg/mL, but the dichloromethane extract and physalin D had low activity against fungi at MICs ranging from 256 to 512 microg/mL. Additionally, the antioxidant activity of physalin D was evaluated by qualitative DPPH (1,1-diphenyl-2-picrylhydrazyl) radical and TBA (thiobarbituric acid) assays. Physalin D showed low antioxidant activity with an IC(50) value of >or= 10 +/- 2.1.

Expression of radial glial markers (GFAP, BLBP and GS) during telencephalic development in the catshark (Scyliorhinus canicula).

Radial glial cells (RGCs) are the first cell populations of glial nature to appear during brain ontogeny. They act as primary progenitor (stem) cells as well as a scaffold for neuronal migration. The proliferative capacity of these cells, both in development and in adulthood, has been subject of interest during past decades. In contrast with mammals where RGCs are restricted to specific ventricular areas in the adult brain, RGCs are the predominant glial element in fishes. However, developmental studies on the RGCs of cartilaginous fishes are scant. We have studied the expression patterns of RGCs markers including glial fibrillary acidic protein (GFAP), brain lipid binding protein (BLBP), and glutamine synthase (GS) in the telencephalic hemispheres of catshark (Scyliorhinus canicula) from early embryos to post-hatch juveniles. GFAP, BLBP and GS are first detected, respectively, in early, intermediate and late embryos. Expression of these glial markers was observed in cells with radial glia morphology lining the telencephalic ventricles, as well as in their radial processes and endfeet at the pial surface and their expression continue in ependymal cells (or tanycytes) in early juveniles. In addition, BLBP- and GS-immunoreactive cells morphologically resembling oligodendrocytes were observed. In late embryos, most of the GFAP- and BLBP-positive RGCs also coexpress GS and show proliferative activity. Our results indicate the existence of different proliferating subpopulations of RGCs in the embryonic ventricular zone of catshark. Further investigations are needed to determine whether these proliferative RGCs could act as neurogenic and/or gliogenic precursors.

Characterization of covalent DNA binding of morpholino and cyanomorpholino derivatives of doxorubicin.

BACKGROUND: The doxorubicin analogues cyanomorpholino doxorubicin (MRA-CN) and morpholino doxorubicin (MRA) were synthesized in an attempt to avoid the cardiotoxicity and drug resistance of doxorubicin therapy. MRA-CN forms interstrand DNA cross-links without requiring microsomal metabolic activation in the presence of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) to form a product that alkylates DNA, but MRA requires metabolic activation. Alkylation produces DNA cross-links, which are associated with potentiation of the cytotoxicity of some drugs. PURPOSE: Our purpose was to study the DNA binding of MRA-CN and MRA with and without metabolic activation in order to better understand the mechanisms for cross-linking DNA. METHODS: We used [3H]MRA and [3H]MRA-CN, with the 3H labeled at C-2 and C-6 of the morpholino ring. MRA (10 nM) was incubated with human liver microsomes with or without NADPH to measure DNA binding. In addition, a filter elution assay was used to determine the nature and extent of drug binding to DNA in the human ovarian carcinoma cell line ES-2. We studied the appearance of interstrand cross-links versus total DNA adducts in pBR322 plasmid DNA incubated with 100 nM MRA-CN in cell-free medium and then subjected to denaturation and agarose gel electrophoresis. RESULTS: Regardless of the extracellular concentration of the drug (1-100 nM), 85% of intracellular MRA-CN was covalently bound to DNA, and the total amount of drug bound to DNA was proportional to extracellular drug concentration. No covalent binding of MRA to DNA was found in cells exposed to 10 nM MRA alone for 2 hours. In contrast, 10% of the intracellular drug was bound to DNA if the cells were exposed to MRA preincubated with human liver microsomes and NADPH. The percentage of plasmids containing at least one interstrand cross-link rose from 35% at 15 minutes to 92% at 2 hours. We estimate that eight molecules of MRA-CN were adducted per molecule of pBR322 DNA (or one drug adduct per 545 base pairs), with a minimum of 12% of the adducts forming interstrand cross-links. CONCLUSIONS: These results suggest that the carbons at positions 2 and 6 of the morpholino ring of both MRA-CN and the activated metabolite of MRA are retained in the drug-DNA adduct. They also indicate that the formation of interstrand DNA cross-links by MRA-CN is preceded by formation of drug adducts to a single strand of DNA.

Mutation rates and mechanisms of resistance to etoposide determined from fluctuation analysis.

BACKGROUND: The major known mechanisms of resistance to etoposide include altered expression of its target enzyme, topoisomerase II (Topo II), and the multidrug-resistant phenotypes encoded by the mdr1 and MRP (multidrug resistance-associated protein) genes. There is little information regarding the distribution, frequency, and origin of these mechanisms in cancer cells. PURPOSE: We performed fluctuation analysis experiments with the human sarcoma cell line, MES-SA, to assess 1) if selection or induction mechanisms are involved in resistance to etoposide, 2) mutation rates for cellular resistance to etoposide, and 3) the nature of the single-step selected surviving clones. METHODS: Three groups of 10 flasks were seeded with more than 2000 cells each and allowed to grow to near confluence (approximately 3 x 10(6) cells per flask). After reseeding, each group received etoposide for 1 week at a final concentration of 0.5 microM (group A), 1.0 microM (group B), and 5.0 microM (group C). Surviving colonies in each of the 30 populations were scored and individually harvested. RESULTS: Mutation rates were estimated at 2.9 x 10(-6) (group A), 5.7 x 10(-7) (group B), and 1.7 x 10(-7) (group C) per cell generation. Of 61 propagated colonies, four of 26 from group A, five of 19 from group B, and none of 16 from group C were stably resistant. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction, conferring etoposide resistance in groups A and B. Five of the stably resistant clones were cross-resistant to doxorubicin. Analysis by polymerase chain reaction failed to detect the expression of the multidrug-resistant gene mdr1 messenger RNA (mRNA) in any of the clones. No increase in expression of the MRP gene was observed. However, a significant decrease in both Topo II alpha and II beta mRNA (30%-70%) was found in six of seven stably resistant and six of six unstably resistant mutants. CONCLUSIONS: Our study demonstrates that resistance to etoposide arises spontaneously, with most clones surviving either stochastically or through very labile mechanisms of resistance. The experimental design has derived a set of resistant mutants from a single-step selection. In those clones, decreased expression of Topo II is the predominant mechanism selected. IMPLICATIONS: These findings suggest that stable resistance to etoposide chemotherapy may be acquired by selection of spontaneously arising mutants rather than induction by drug exposure. The stably resistant clones may represent descendants from a single mutational event in each population.

Decreased mutation rate for cellular resistance to doxorubicin and suppression of mdr1 gene activation by the cyclosporin PSC 833.

BACKGROUND: Various mechanisms can contribute to cellular resistance to doxorubicin. These include expression of the multidrug transporter P-glycoprotein (product of the mdr1 gene [also known as PGY1], Mrp (multidrug resistance-associated protein), the p110 major vault protein, altered glutathione metabolism, and altered levels or activity of topoisomerase II (Topo II). We reported recently that single-step treatment of human MES-SA sarcoma cells with 40 nM doxorubicin resulted in selection of spontaneous mutants at a rate of 1.8 x 10(-6) per cell generation. All individually selected mutants manifested the multidrug-resistant phenotype, related to activation of the mdr1 gene. PURPOSE: Luria and Delbrück fluctuation analysis was performed with MES-SA cells to determine the mutation rate and the nature and mechanisms of resistance after single-step selection with doxorubicin in the presence of the cyclosporin PSC 833, a potent modulator of multidrug resistance. METHODS: Ten flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 8 x 10(6) cells. After reseeding in 96-well plates, the populations were treated with 40 nM doxorubicin and 2 microM PSC 833 for 3 weeks. Surviving colonies were scored, individually harvested, and propagated. The drug-resistant phenotype was assessed by the tetrazolium dye (MTT) cytotoxicity assay and by monitoring cellular glutathione content and radiolabeled drug accumulation. Coupled reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate mdr1, MRP, Topo II alpha, and Topo II beta gene expression. Topo II, P-glycoprotein, and p110 levels were examined by immunoblotting or immunocytochemistry. Topo II activity was assessed by decatenation of kinetoplast DNA, and etoposide-induced cleavable complex formation was studied by the potassium-sodium dodecyl sulfate precipitation assay. RESULTS: Mutations were detected at a rate of 2.5 x 10(-7) per cell generation. Analysis of variance indicates that spontaneous mutations, rather than changes in cellular function, conferred resistance to doxorubicin and PSC 833. None of the isolated clones expressed mdr1 messenger RNA or P-glycoprotein, and none exhibited an increase in MRP expression. No alterations were found in cellular glutathione content, intracellular accumulations of daunorubicin and etoposide, levels of p110 protein, or levels of Topo II beta transcripts. However, a significant decrease in Topo II alpha messenger RNA and protein was found in all examined clones, as well as decreased Topo II catalytic activity and reduced cleavable complex formation in the presence of etoposide. CONCLUSIONS: PSC 833 co-selection reduced the mutation rate for doxorubicin-selected resistance by 10-fold and suppressed the emergence of mdr1 mutants. Survival of cells exposed to doxorubicin and PSC 833 occurs by selection of spontaneously arising mutants that exhibit altered Topo II alpha expression. IMPLICATIONS: Our results suggest that treatment with multidrug resistance modulators such as PSC 833 together with multidrug resistance-related cytotoxins may suppress the activation of mdr1 and prevent the emergence of resistant cancer cell clones with the multidrug-resistant phenotype.

Prevalence of multidrug resistance related to activation of the mdr1 gene in human sarcoma mutants derived by single-step doxorubicin selection.

Fluctuation analysis experiments were performed in the human sarcoma cell line MES-SA to assess whether selection or induction mechanisms determine resistance to doxorubicin (DOX), mutation rates, and the nature of the surviving clones. Thirteen flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 3.3 x 10(6) cells. After reseeding in 96-well plates, each population was treated with 40 nM DOX for 2 weeks. Surviving colonies were scored and harvested. Clones were propagated and analyzed for drug resistance phenotype. Expression of the mdr1, mrp, and topoisomerase II alpha and II beta genes was analyzed by reverse transcription-polymerase chain reaction. Accumulation of the P-glycoprotein substrate rhodamine-123 was measured by flow cytometry, with and without the cyclosporin D analogue SDZ PSC 833. Cellular glutathione levels were measured by flow cytometry, and M(r) 110,000 vesicular protein (p110) expression was detected by immunohistochemistry. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction conferring DOX resistance. At this stringent level (5-6 log cell killing) of drug exposure, the mutation rate was estimated at 1.8 x 10(-6) per cell generation. All 30 propagated clones demonstrated cross-resistance to vinblastine, etoposide, and paclitaxel (Taxol), but not to cisplatin or bleomycin. Increased mRNA levels of mdr1 were observed in all 27 clones tested, including at least 1 from each of the 13 populations. No alterations were found in expression or level of topoisomerase II alpha or II beta, mrp, glutathione, and p110. Expression of P-glycoprotein was confirmed by flow cytometry using the monoclonal antibody UIC2. In almost all tested clones, decreased intracellular rhodamine-123 accumulation was modulated by 2 microM SDZ PSC 833, and the vinblastine resistance in all examined clones was completely reversed by SDZ PSC 833 and verapamil. Our study demonstrates that survival of cells exposed to DOX in a single step occurs as a result of a stochastic process consistent with mutational events. Activation of the mdr1 gene is the predominant mechanism selected by DOX in these resistant clones.

Role of cytochrome P-450 from the human CYP3A gene family in the potentiation of morpholino doxorubicin by human liver microsomes.

The cytotoxicity of the morpholino derivative of doxorubicin (MRA) can be potentiated 50- to 100-fold by human liver microsomes and NADPH (J. Natl. Cancer Inst., 81: 1034, 1989). This metabolic potentiation is inhibited by carbon monoxide or hypoxia, indicating that it is cytochrome P-450-dependent. The potentiation is also inhibited by the cytochrome P-450 inhibitors, SKF-525A and cimetidine. The metabolism by the microsomes is substrate-specific, varying markedly with alterations of either the morpholino or anthracycline ring substituents. No potentiation occurred with doxorubicin itself, or the cyanomorpholinyl, methoxypiperidinyl, N-hydroxyethyl or the O-bridged cyanomorpholinyl analogues of doxorubicin. We utilized a panel of human liver microsomes and cytochrome P-450 type-specific antibodies to further identify the isoform(s) of cytochrome P-450 that potentiated the cytotoxicity of MRA. The potentiation correlates well with the benzyloxyresorufin assay (r2 = 0.98) and aflatoxin B1 metabolism (r2 = 0.98), both assays that are relatively specific for CYP3A proteins. Correlations were also observed for the expression of protein(s) cross-reacting with an antibody against rat cytochrome P-450 CYP3A1 (r2 = 0.97) and MRA metabolism. This antibody against the rat cytochrome P-450 CYP3A isoform(s) inhibited more than 90% of the potentiation of the cytotoxicity by human liver microsomes. Antibodies against the CYP1A2, CYP2C6, and CYP2B2 isoforms produced no inhibition, nor did their expression by Western blotting correlate with MRA potentiation. Complete inhibition of the potentiation of MRA by human liver microsomes was found when the CYP3A substrates cyclosporin A and erythromycin were used in the reaction system. These data indicate that the CYP3A isoform(s) of cytochrome P-450 play a major role in the metabolism of MRA in vitro to a more active species.

Resistance mechanisms in human sarcoma mutants derived by single-step exposure to paclitaxel (Taxol).

A fluctuation analysis experiment was performed by exposing 15 expanded populations of MES-SA sarcoma cells to paclitaxel (Taxol) at a concentration of 10 nM for 7 days. The mutation rate was approximately 8 multiplied by 10(-7)/cell generation. ANOVA supports a stochastic cell survival mechanism of spontaneous mutation rather than induction of an adaptive response under these selection conditions. Surviving colonies were found in 12 populations, 9 of which had clones that remained resistant to paclitaxel after a 2-month period of propagation. Analysis of mdr1 gene expression by reverse transcription PCR demonstrated positive clones in 4 of the 9 populations with stable resistance. Accumulation of [(3)H]paclitaxel was decreased in these clones but not in the mdr1-negative clones compared with parental cells. A high degree of resistance to paclitaxel (36- to 93-fold) was selected by this single drug exposure in all 9 stably resistant mutants. Those with mdr1 activation demonstrated a broad cross-resistance to vinblastine, doxorubicin, and etoposide, whereas the other 6 mutants were cross-resistant only to the Vinca alkaloids. Because tubulins are the target molecules for paclitaxel cytotoxicity, we evaluated total tubulin content by immunoblotting and performed semiquantitative reverse transcription PCR analysis for expression of the alpha-tubulin isotypes B alpha 1, K alpha 1 and H alpha 44, the beta-tubulin isotypes M40, beta9, 5beta, beta2 and beta4, and gamma-tubulin. Total tubulin content was decreased significantly in one of the single-step mutants. All surviving clones, both resistant and sensitive to paclitaxel, displayed reduced expression of the 5beta and beta 4 beta-tubulin isotype transcripts in comparison with the parental cell line. These data suggest that stringent exposure to paclitaxel selected clones with reduced transcript levels of 5beta and beta4 beta-tubulin isotypes, but that these reduced levels were not directly involved in the resistance of the clones to paclitaxel. The results suggest an important role for non-multidrug-resistant mechanisms of resistance to paclitaxel. These mechanisms do not involve reduced drug accumulation and provide cross-resistance among both paclitaxel and tubulin depolymerizing agents.

Volume-activated chloride current is not related to P-glycoprotein overexpression.

It has been suggested that P-glycoprotein (P-gp), an ATP-dependent transporter responsible for classical multidrug resistance, is also a volume-regulated chloride channel. We reexamined this hypothesis by use of whole-cell patch clamp recordings of three matched pairs of cell lines, which were either drug-sensitive or drug-resistant due to P-gp overexpression. We demonstrate here that volume-regulated chloride-selective currents can be induced in cells with or without P-gp expression. Overexpression of either P-gp or cystic fibrosis transmembrane conductance regulator, the protein product of the CF gene and another member of the ATP-dependent transporters, is associated with a hypotonicity-induced, rapid onset, transient current prior to onset of the volume-sensitive chloride-selective current, an apparent nonspecific effect related to the overexpression of an integral membrane protein. These results suggest that there is no relationship between P-gp and the chloride channel activated by cell swelling.

Inhibition of lysosomal acid sphingomyelinase by agents which reverse multidrug resistance.

An increasing body of evidence appears to implicate the lipid bilayer of multidrug resistant (MDR) cells with P-glycoprotein activity. Several cationic amphiphilic drugs (CADs) have been extensively described as modulators of MDR. These same agents are also known to (1) inhibit lysosomal acid sphingomyelinase (ASmase), a phospholipid degrading enzyme, and/or (2) induce phospholipidosis in animal tissues or cultured cell lines. In this report, we randomly selected 17 CADs and evaluated their potency in modulating MDR in the murine MDR P388/ADR leukemia cell line. We compared these results with their ability to inhibit ASmase and observed a significant dose-dependent linear relationship (95% central confidence interval), between ASmase inhibition and MDR reversal. This approach permitted us to identify three new modestly potent chemosensitizers: trimipramine, desipramine, and mianserine. Modulation of MDR was not cell line specific, since CADs at 10 microM increased doxorubicin (DOX) and vinblastine (VBL) (but not methotrexate, MTX) cytotoxicity in both P388/ADR and the human MDR cell lines MES-SA/Dx5 and K562/R7, but not in the parental drug-sensitive cells. Although all chemosensitizing CADs at 10 microM significantly increased Rhodamine-123 (Rho-123) accumulation in the human leukemia MDR cell line K562/R7 and most presented significant displacement of the photoaffinity labelling probe iodoarylazidoprazosin, no correlation between these observations and the ability of CADs to sensitize MDR cells to DOX and VBL was found. In conclusion, our study strongly suggests that the chemosensitizing potency of agents such as CADs may be due to a dual mechanism of action: direct antagonism of P-gp activity and indirect modulation of P-gp activity through the disruption of cellular lipid metabolism.