Glutathione S-transferase does not play a role in multidrug resistance of L1210/VCR cell line

Multidrug resistance of cancer cells is often accompanied by the (over)expression of integral plasma membrane P-glycoprotein, an ATP-dependent transport pump for diverse unrelated compounds. The glutathione detoxification system represents another mechanism that may be involved in multidrug resistance. In the multidrug-resistant L1210/VCR cell line obtained by long-term adaptation of parental L1210 cells to vincristine, an increased expression of P-glycoprotein has previously been established. In this paper, we investigated if the glutathione detoxification system is also involved in the multidrug resistance of these cells. L1210/VCR cells with resistance induced by adaptation to vincristine were also found to be cross-resistant to vinblastine, actinomycin D, mitomycin C, doxorubicin and cyclophosphamide. The resistance of the above cells to vincristine and doxorubicin was accompanied by a depression of drug accumulation (which has not yet been established for other drug). L1210/VCR cells are able to survive better than sensitive cells under conditions when glutathione was depleted by L-buthionine sulfoximine. Nevertheless, L-buthionine sulfoximine did not influence the resistance of L1210/VCR cells to vincristine. Moreover, the presence of sublethal concentrations of cytostatics neither changed the IC50 value of resistant cells to L-buthionine sulfoximine nor the cytoplasmic activity of glutathione S-transferase, the crucial enzyme of glutathione detoxification system. All the above findings indicate that the glutathione detoxification system is not involved in the mechanisms that ensure the multidrug resistance phenotype of L1210/VCR cells.     

CD36 does not play a direct role in HDL or LDL metabolism

CD36 and scavenger receptor class B, type I (SR-BI) are both class B scavenger receptors that recognize a broad variety of ligands, including oxidized low density lipoprotein (oxLDL), HDL, anionic phospholipids, and apoptotic cells. In this study we investigated the role of mouse CD36 (mCD36) as a physiological lipoprotein receptor. We compared the association of various lipoprotein particles with mCD36 and mSR-BI expressed in COS cells by adenovirus-mediated gene transfer. mCD36 bound human oxLDL and mouse HDL with high affinity. Human LDL bound poorly to mCD36, indicating that mCD36 is unlikely to play a significant role in LDL metabolism. The ability of mCD36 to mediate the selective uptake of cholesteryl esters (CE) from receptor-bound HDL was assessed. In comparison with mSR-BI, mCD36 inefficiently mediated the selective uptake of CE. Hepatic overexpression of mCD36 in C57BL/6 mice by adenovirus-mediated gene transfer did not result in significant alterations in plasma LDL and HDL levels. We conclude that mCD36, while able to bind HDL with high affinity, does not contribute significantly to HDL or LDL metabolism.     

Validation of a blood protein signature for non-small cell lung cancer

Background

CT screening for lung cancer is effective in reducing mortality, but there are areas of concern, including a positive predictive value of 4% and development of interval cancers. A blood test that could manage these limitations would be useful, but development of such tests has been impaired by variations in blood collection that may lead to poor reproducibility across populations.

Results

Blood-based proteomic profiles were generated with SOMAscan technology, which measured 1033 proteins. First, preanalytic variability was evaluated with Sample Mapping Vectors (SMV), which are panels of proteins that detect confounders in protein levels related to sample collection. A subset of well collected serum samples not influenced by preanalytic variability was selected for discovery of lung cancer biomarkers. The impact of sample collection variation on these candidate markers was tested in the subset of samples with higher SMV scores so that the most robust markers could be used to create disease classifiers. The discovery sample set (n = 363) was from a multi-center study of 94 non-small cell lung cancer (NSCLC) cases and 269 long-term smokers and benign pulmonary nodule controls. The analysis resulted in a 7-marker panel with an AUC of 0.85 for all cases (68% adenocarcinoma, 32% squamous) and an AUC of 0.93 for squamous cell carcinoma in particular. This panel was validated by making blinded predictions in two independent cohorts (n = 138 in the first validation and n = 135 in the second). The model was recalibrated for a panel format prior to unblinding the second cohort. The AUCs overall were 0.81 and 0.77, and for squamous cell tumors alone were 0.89 and 0.87. The estimated negative predictive value for a 15% disease prevalence was 93% overall and 99% for squamous lung tumors. The proteins in the classifier function in destruction of the extracellular matrix, metabolic homeostasis and inflammation.

Conclusions

Selecting biomarkers resistant to sample processing variation led to robust lung cancer biomarkers that performed consistently in independent validations. They form a sensitive signature for detection of lung cancer, especially squamous cell histology. This non-invasive test could be used to improve the positive predictive value of CT screening, with the potential to avoid invasive evaluation of nonmalignant pulmonary nodules.     

Sustained metaphase arrest in response to ionizing radiation in a non-small cell lung cancer cell line

Kodym, E., Kodym, R., Choy, H. and Saha, D. Sustained Metaphase Arrest in Response to Ionizing Radiation in a Non-small Cell Lung Cancer Cell Line. Radiat. Res. 169, 46-58 (2008). In solid tumors, non-apoptotic forms of tumor cell inactivation such as mitotic catastrophe appear to be predominant in the response to DNA-damaging agents. Despite its importance, the underlying molecular mechanisms of mitotic catastrophe have been only partially elucidated. We found that a large fraction of HCC2279 non-small cell lung cancer cells underwent mitotic catastrophe after irradiation. Cells were arrested in metaphase with chromosomal damage indicated by DNA fragments displaced from the metaphase plate and considerable numbers of residual gamma-H2AX foci. Although TP53 was nonfunctional, we detected a prompt radiation response on the level of checkpoint kinases. In contrast, CDC25A was the only checkpoint phosphatase that was responsive to radiation. CDC25B was not detectable, and CDC25C was constitutively phosphorylated at serine 216, leading to its cytoplasmic sequestration and functional inactivation. Therefore, radiation-induced mitotic catastrophe in HCC2279 cells appears to be induced by a combination of relative insufficiencies in the p53-mediated and checkpoint kinase-mediated pathways leading to premature entry into mitosis. Displaced chromosome fragments triggering an intra-M checkpoint in cells entering mitosis presumably result in a sustained metaphase arrest. The phenomenon found in these cells, which were derived directly from a human patient, might be responsible for therapy-induced genetic instability of tumors.     

Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

Drug dosing is commonly based on the dogma that, increasing the dose maximizes the therapeutic response until a dose level that is prohibitively toxic is reached. This doctrine also applies to antibody therapy, as several protocols have explored dose escalation. We have analyzed the effect of different amounts of a homophilic Herceptin targeting a human lung tumor cell line, and discovered that the normal dose-potency relationship does not apply. To study this paradoxical effect of antibody concentration on potency, we examined the molecular species of the homophilic Herceptin under different concentrations using size exclusion chromatography and gel electrophoresis. We also varied experimental conditions in FACS tumor targeting, such as concentration of antibody, membrane immobilization, temperature, and antibody homo/dimer immobilization. We observed that high concentrations of homophilic Herceptin reduce targeting, and also noted the tumor growth arrest in the xenograft mice after the tumor reached a critical size. The therapeutic window appears to be defined by tumor size and antibody concentration. Since the concentration of this homophilic antibody defines the optimum targeting window, our data suggest the therapeutic dose of antibody should be matched with the tumor burden.     

X-ray irradiation altered chemosensitivity of a p53-null non-small cell lung cancer cell line

Radiotherapy is an effective approach to treating many types of cancer. Recent progress in radiotherapy technology, such as intensity-modulated radiation therapy (IMRT) and three-dimensional (3D) radiotherapy, allow precise energy transfer to the tumor, which has improved local control rates. However, the emergence of tolerant cells during or after radiotherapy remains problematic. In the present study, we first established a cell population from H1299, the p53-null non-small cell lung cancer cell line, by 10 Gy irradiation using 6 MV X-rays. The radio- and chemosensitivity of this cell population (referred to as H1299-IR) was determined using colony formation analyses and MTS assays. Compared with the parental cell line, the radiosensitivity of H1299-IR was apparently the same. H1299 and H1299-IR were both more radio tolerant than the A549 cell line. However, H1299-IR became significantly more sensitive to cisplatin, an antitumor agent. After exposure to 25 mug/ml cisplatin for 2 h, parental cells steadily grew during the MTS assay, whereas the sensitivity of H1299-IR cells doubled both at 24 and 48 h. Microarray analysis of over 30,000 H1299-IR genes (Agilent Technology) revealed that 12 and 15 genes were up- (> 2.0) and down- (< 2.0) regulated, respectively. Rad51d (homologous recombination repair protein) gene was down-regulated 2.8-fold, whereas matrix metalloproteinase 1 (collagenase-1) gene was up-regulated 4.4-fold. These results indicated that some p53-null non-small cell lung cancers could be successfully treated when X-ray radiotherapy was administered with subsequent or concurrent cisplatin chemotherapy.     

UPregulated single-stranded DNA-binding protein 1 induces cell chemoresistance to cisplatin in lung cancer cell lines

In order to understand the molecular basis of cold adaptation, we have used directed evolution to transform a thermophilic lipase LipR1 into its psychrophilic counterpart. A single round of random mutagenesis followed by screening for improved variants yielded a mutant with single-point mutation LipR1M1 (S130T), with optimum activity at 20?°C. Its activity at 50?°C is only 20% as compared to wild type (100%). It showed catalytic rate constant (k _cat) 3 times higher and a catalytic efficiency (k _cat/K _m) 4 times that of wild type. Circular dichroism and fluorescence studies also supported our observation of mutant structural flexibility. Structure analysis using homology models showed that Threonine 130 is exposed to solvent and has lost H-bond interaction with neighboring amino acid, thereby increasing flexibility of this lipase structure.     

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, "the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance", has instigated lots of debates. In this study, according to the previous research of PprI’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. ra-diodurans R1 strain, pprI function-deficient mutant (YR1), and pprI function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence mi-croscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells ex-hibit the tightly packed ring-like morphology, while the pprI function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprI completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprI partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a PprI C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensi-tive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.     

5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis

Background  

Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC.     

Target volume definition for staple line recurrences of non-small cell lung cancer

BackgroundStaple line (SL) recurrences of non-small cell lung cancer (NSCLC) are commonly treated with radiotherapy (RT), but the target volume definition — whole SL versus focused on recurrence — is unclear. The aim of the study was to determine the appropriate target volume for RT of SL recurrences.Materials and methodsTwenty-two consecutive patients (20 stage I, 2 stage II) treated with salvage RT for SL recurrences were retrospectively analyzed. Imaging features at the time of SL recurrence were evaluated to guide target volume definition.ResultsSurgeryAll patients had complete tumor resection (wedge resection in 10 (45%) and lobectomy in 12 (55%) patients). 14 (64%) patients had risk factors for recurrence, including surgical margins ≤ 2 cm, angiolymphatic and visceral pleural invasion.Salvage RTAfter a median 26 months (9–67), all 22 patients developed SL recurrence which was metabolically active on PET in all and biopsy-confirmed in 18/22 (82%) patients. All patients underwent RT targeting the location of the SL recurrence only. 13/22 (59%) patients had additional PE T-negative nodular or linear SL changes that were not included in the irradiated volume.Recurrence after RTAfter a median 17 months (9–34) 10/22 (45%) patients recurred either regionally 6/10 (60%), in the lungs 4/10 (40%) or distally 3/10 (30%). No patient recurred at the SL. Two-year overall and disease-free survival rates after RT were 71% and 65%, respectively.ConclusionRT to SL recurrences alone results in excellent local control. Additional treatment to reduce regional and distant recurrences should be considered.     

Activated armadillo/beta-catenin does not play a general role in cell migration and process extension in Drosophila.

Human beta-catenin and its fly homolog Armadillo are best known for their roles in cadherin-based cell-cell adhesion and in transduction of Wingless/Wnt signals. It has been hypothesized that beta-catenin may also regulate cell migration and cell shape changes, possibly by regulating the microtubule cytoskeleton via interactions with APC. This hypothesis was based on experiments in which a hyperstable mutant form of beta-catenin was expressed in MDCK cells, where it altered their migratory properties and their ability to send out long cellular processes. We tested the generality of this hypothesis in vivo in Drosophila. We utilized three model systems in which cell migration and/or process extension are known to play key roles during development: the migration of the border cells during oogenesis, the extension of axons in the nervous system, and the migration and cell process extension of tracheal cells. In all cases, cells expressing activated Armadillo were able to migrate and extend cell processes essentially normally. The one alteration from normal involved an apparent cell fate change in certain tracheal cells. These results suggest that only certain cells are affected by activation of Armadillo/beta-catenin, and that Armadillo/beta-catenin does not play a general role in inhibiting cell migration or process extension.     

Apoptosis in non-small cell lung cancer as related to drug resistance and prognosis

The percentage of apoptotic cells among the tumor cells (apoptotic index) was determined in a series of 178 non-small cell lung carcinomas with a long term clinical follow-up by a terminal desoxynucleotidyl transferase mediated dUTP nick end labelling technique. In survival analysis, we found no statistically significant correlation between the apoptotic index and survival times. We estimated also the sensitivity of specimens to doxorubicin by a short term test. Tumors with a high apoptotic activity were more sensitive to doxorubicin than tumors with a less apoptotic index. Thus, our data indicate that apoptosis may be involved in drug response of lung tumors and it could be useful for the chemotherapeutic strategy to design drugs which trigger apoptosis.     

h-ANF does not play a role in the regulation of myocardial force of contraction

In the present study, h-ANF failed to reduce myocardial force of contraction alone and in the presence of isoprenaline. This lack of effect was observed in electrically driven, isolated human papillary muscle strips and rat papillary muscles. In contrast, adenosine and carbachol exerted antiadrenergic actions in rat and human myocardium. It is concluded that h-ANF obviously does not play a role in the regulation of human and rat myocardial force of contraction, whereas adenosine and carbachol are capable to produce antiadrenergic effects in the human and rat myocardium.     

USP52 inhibits cell proliferation by stabilizing PTEN protein in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer. Ubiquitination is closely related to the development of lung cancer. However, the biological importance of newly discovered ubiquitin-specific peptidase (USP) 52 (USP52) in NSCLC remained unclear. Here, our findings identify USP52 as a novel tumor suppressor of NSCLC, the low expression of USP52 predicts a poor prognosis for NSCLC patients. The present study demonstrates that USP52 inhibits cancer cell proliferation through down-regulation of cyclin D1 (CCND1) as well as AKT/mTOR signaling pathway inhibition. Meanwhile, USP25 also suppresses NSCLC progression via enhancing phosphatase and tensin homolog (PTEN) stability in cancer cells, which further indicates the significance/importance of USP52 in NSCLC suppression.     

The role of adjuvant chemotherapy in the treatment of non-small cell lung cancer

Postoperative treatments for lung cancer have been evaluated for more than two decades, but in the majority of the studies (especially until 1990) no significant effect on survival has been shown. In 1995, a meta-analysis of eight cisplatin-based adjuvant chemotherapy trials with NSCLC showed a 13% reduction in the risk of death (P=0.08) and 5% benefit in 5-year survival. Among four positive trials (IALT, JBR10, ANITA and CALGB study) the absolute increase in the 5-year survival rates by adjuvant chemotherapy ranged from 4% to 15%, and the hazard ratios for death ranged from 0.6 to 0.86. The author analyzes the most important studies. Conclusion: adjuvant chemotherapy after complete resection of NSCLC can be considered as a new standard of care in patients with good performance status. Patients should receive 4 cycles of platina-based chemotherapy beginning 4-8 weeks after surgery. Future perspectives: optimization of chemotherapy regimens including targeted therapy (such as EGFR inhibitors, angiogenesis inhibitors, anti-EGFR monoclonal antibodies). Further progress is anticipated through the integration of chemopreventive agents into the adjuvant treatment.     

Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products.     

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, “the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance”, has instigated lots of debates. In this study, according to the previous research of Pprl’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. radiodurans R1 strain, pprf function-deficient mutant (YR1), and pprl function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence microscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells exhibit the tightly packed ring-like morphology, while the pprl function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprl completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprl partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a Pprl C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensitive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance. Supported by the National Basic Research Program of China (Grant No. 2004CB19604), the National Natural Science Foundation of China (Grant No. 30330020), and the National Science fund for Distinguished Young Scholars (Grant No. 30425038)