Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Contribution of PGAP3 co-amplified and co-overexpressed with ERBB2 at 17q12 involved poor prognosis in gastric cancer.

The locus at 17q12 erb-b2 receptor tyrosine kinase 2 (ERBB2) has been heavily amplificated and overexpressed in gastric cancer (GC), but it remains to be elucidated about the clinical significance of the co-amplification and co-overexpression of PGAP3 gene located around ERBB2 in GC. The profile of PGAP3 and ERBB2 in four GC cell lines and tissue microarrays containing 418 primary GC tissues was assessed to investigate the co-overexpression and clinical significance of the co-amplified genes, and to evaluate the impact of the co-amplified genes on the malignancy of GC. Co-amplification of PGAP3 and ERBB2 accompanied with co-overexpression was observed in a haploid chromosome 17 of NCI-N87 cells with double minutes (DMs). PGAP3 and ERBB2 were overexpressed and positively correlated in 418 GC patients. Co-overexpression of the PGAP3 and ERBB2 was correlated with T stage, TNM stage, tumour size, intestinal histological type and poor survival proportion in 141 GC patients. In vitro, knockdown of the endogenous PGAP3 or ERBB2 decreased cell proliferation and invasion, increased G1 phase accumulation and induced apoptosis in NCI-N87 cells. Furthermore, combined silencing of PGAP3 and ERBB2 showed an additive effect on resisting proliferation of NCI-N87 cells compared with targeting ERBB2 or PGAP3 alone. Taken together, the co-overexpression of PGAP3 and ERBB2 may be crucial due to its significant correlation with clinicopathological factors of GC. Haploid gain of PGAP3 co-amplified with ERBB2 is sufficient to facilitate the malignancy and progression of GC cells in a synergistic way.

Inhibiting homologous recombination decreases extrachromosomal amplification but has no effect on intrachromosomal amplification in methotrexate-resistant colon cancer cells.

Gene amplification, which involves the two major topographical structures double minutes (DMs) and homegeneously stained region (HSR), is a common mechanism of treatment resistance in cancer and is initiated by DNA double-strand breaks. NHEJ, one of DSB repair pathways, is involved in gene amplification as we demonstrated previously. However, the involvement of homologous recombination, another DSB repair pathway, in gene amplification remains to be explored. To better understand the association between HR and gene amplification, we detected HR activity in DM- and HSR-containing MTX-resistant HT-29 colon cancer cells. In DM-containing MTX-resistant cells, we found increased homologous recombination activity compared with that in MTX-sensitive cells. Therefore, we suppressed HR activity by silencing BRCA1, the key player in the HR pathway. The attenuation of HR activity decreased the numbers of DMs and DM-form amplified gene copies and increased the exclusion of micronuclei and nuclear buds that contained DM-form amplification; these changes were accompanied by cell cycle acceleration and increased MTX sensitivity. In contrast, BRCA1 silencing did not influence the number of amplified genes and MTX sensitivity in HSR-containing MTX-resistant cells. In conclusion, our results suggest that the HR pathway plays different roles in extrachromosomal and intrachromosomal gene amplification and may be a new target to improve chemotherapeutic outcome by decreasing extrachromosomal amplification in cancer.

Sensor Drift Compensation Based on the Improved LSTM and SVM Multi-Class Ensemble Learning Models.

Drift is an important issue that impairs the reliability of sensors, especially in gas sensors. The conventional method usually adopts the reference gas to compensate for the drift. However, its classification accuracy is not high. We propose a supervised learning algorithm that is based on multi-classifier integration for drift compensation in this paper, which incorporates drift compensation into the classification process, motivated by the fact that the goal of drift compensation is to improve the classification performance. In our method, with the obtained characteristics of sensors and the advantage of Support Vector Machine (SVM) in few-shot classification, the improved Long Shot Term Memory (LSTM) is integrated to build the multi-class classifier model. We tested the proposed approach on the publicly available time series dataset that was collected over three years by the metal-oxide gas sensors. The results clearly indicate the superiority of multiple classifier approach, which achieves higher classification accuracy as compared with different approaches during testing period with an ensemble of classifiers in the presence of sensor drift over time.

Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells.

Double minute chromosomes (DMs) are extrachromosomal cytogenetic structures found in tumour cells. As hallmarks of gene amplification, DMs often carry oncogenes and drug-resistance genes and play important roles in malignant tumour progression and drug resistance. The mitogen-activated protein kinase (MAPK) signalling pathway is frequently dysregulated in human malignant tumours, which induces genomic instability, but it remains unclear whether a close relationship exists between MAPK signalling and DMs. In the present study, we focused on three major components of MAPK signalling, ERK1/2, JNK1/2/3 and p38, to investigate the relationship between MAPK and DM production in tumour cells. We found that the constitutive phosphorylation of ERK1/2, but not JNK1/2/3 and p38, was closely associated with DMs in tumour cells. Inhibition of ERK1/2 activation in DM-containing and ERK1/2 constitutively phosphorylated tumour cells was able to markedly decrease the number of DMs, as well as the degree of amplification and expression of DM-carried genes. The mechanism was found to be an increasing tendency of DM DNA to break, become enveloped into micronuclei (MNs) and excluded from the tumour cells during the S/G2 phases of the cell cycle, events that accompanied the reversion of malignant behaviour. Our study reveals a linkage between ERK1/2 activation and DM stability in tumour cells.

Novel role for non-homologous end joining in the formation of double minutes in methotrexate-resistant colon cancer cells.

BACKGROUND: Gene amplification is a frequent manifestation of genomic instability that plays a role in tumour progression and development of drug resistance. It is manifested cytogenetically as extrachromosomal double minutes (DMs) or intrachromosomal homogeneously staining regions (HSRs). To better understand the molecular mechanism by which HSRs and DMs are formed and how they relate to the development of methotrexate (MTX) resistance, we used two model systems of MTX-resistant HT-29 colon cancer cell lines harbouring amplified DHFR primarily in (i) HSRs and (ii) DMs. RESULTS: In DM-containing cells, we found increased expression of non-homologous end joining (NHEJ) proteins. Depletion or inhibition of DNA-PKcs, a key NHEJ protein, caused decreased DHFR amplification, disappearance of DMs, increased formation of micronuclei or nuclear buds, which correlated with the elimination of DHFR, and increased sensitivity to MTX. These findings indicate for the first time that NHEJ plays a specific role in DM formation, and that increased MTX sensitivity of DM-containing cells depleted of DNA-PKcs results from DHFR elimination. Conversely, in HSR-containing cells, we found no significant change in the expression of NHEJ proteins. Depletion of DNA-PKcs had no effect on DHFR amplification and resulted in only a modest increase in sensitivity to MTX. Interestingly, both DM-containing and HSR-containing cells exhibited decreased proliferation upon DNA-PKcs depletion. CONCLUSIONS: We demonstrate a novel specific role for NHEJ in the formation of DMs, but not HSRs, in MTX-resistant cells, and that NHEJ may be targeted for the treatment of MTX-resistant colon cancer.

Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells.

Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, ß-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

Expulsion of micronuclei containing amplified genes contributes to a decrease in double minute chromosomes from malignant tumor cells.

Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM-carried genes remains to be clarified. The human colorectal cancer cell line NCI-H716 and human malignant primitive neuroectodermal tumor cell line SK-PN-DW are known to contain many DMs. To examine the amplification of DM-carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI-H716 and SK-PN-DW tumor cells. We identified the amplification regions and the DM-carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM-carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM-carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM-carried genes led to a decrease in the number of DMs and reduced the amplification of the DM-carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of γH2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM-carried genes resulted in a decrease in cell proliferation and invasion ability.

Effects of deep, air and vacuum frying on oyster quality and protein-mediated mechanism analysis via TMT quantitative proteomics.

Fried oyster is a popular aquatic food product in East Asia, but nutrient loss during thermal processing become a significant concern. The goal of this research was to examine the impact of distinct frying techniques, including deep frying (DF), air frying (AF), and vacuum frying (VF), on the nutritional, textural and flavor characteristics of oysters. The VF method demonstrated superior retention of beneficial properties and flavor, and reduced protein and lipid oxidation compared to the DF and AF methods. Furthermore, proteomic analysis of oysters was attempted to explain the molecular mechanisms governing the influence of key differential proteins. 20 major differential proteins, including actin-2 protein, tryptophan 2,3-dioxygenase and 1-alph, involved in oyster protein oxidation were identified, annotated and analyzed to elucidate their influence mechanisms. This research provides a deeper understanding of intricate interactions between frying techniques and oyster biochemistry, which offers valuable implications for enhancing food quality in seafood industry.

De novo-generated small palindromes are characteristic of amplicon boundary junction of double minutes.

Double minutes (DMs) are hallmarks of gene amplification. However, their molecular structure and the mechanisms of formation are largely unknown. To elucidate the structure and underlying molecular mechanism of DMs, we obtained and cloned DMs using microdissection; and degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR) from the ovarian cancer cell line UACC-1598. Two large amplicons, the 284 kb AmpMYCN, originating from locus 2p24.3 and the 391 kb AmpEIF5A2, from locus 3q26.2, were found co-amplified on the same DMs. The two amplicons are joined through a complex 7 kb junction DNA sequence. Analysis of the junction has revealed three de novo created small palindromes surrounding the six breakpoints. Consistent with these observations, we further found that 70% of the 57 reported DM junction sequences have de novo creation of small palindromic sequences surrounding the breakpoints. Together, our findings indicate that de novo-generated small palindromic sequences are characteristic of amplicon boundary junctions on DMs. It is possible that the de novo-generated small palindromic sequences, which may be generated through non-homologous end joining in concert with a novel DNA repair machinery, play a common role in amplicon rejoining and gene amplification.

Diacerein plus glucosamine hydrochloride improves the safety and efficacy and inhibit inflammatory factors in the treatment of knee osteoarthritis.

OBJECTIVES: The aim of this study is to elucidate the safety and efficacy of diacerein (DIA) plus glucosamine hydrochloride (GlcN·HCl) in the treatment of knee osteoarthritis (KOA) and their effect on inflammatory factors (IFs). METHODS: Retrospectively, 116 KOA patients admitted between August 2018 and August 2021 were selected. Among them, 55 cases received DIA monotherapy (control group, Con) and 61 cases received DIA + GlcN·HCl (observation group, Obs). The efficacy, safety, scores of Lequesne Index, and Visual Analogue Scale (VAS), as well as the levels of IFs of the two groups were observed and compared. Further, Cox regression was used to perform an in-depth analysis of factors influencing the occurrence of complications in patients with KOA. RESULTS: The analyses revealed a higher overall response rate and a lower adverse event rate in the Obs group compared with the Con group, with statistical significance. Decreased scores of Lequesne Index and VAS and levels of IFs were determined in the Obs after treatment, which were all significantly lower compared with those of the Con. Cox regression analysis identified that TNF-α, IL-1ß, hs-CRP, and treatment mode affected the occurrence of complications in KOA patients. CONCLUSIONS: DIA + GlcN·HCl can significantly inhibit the inflammation level in KOA, with definite curative effects and a favorable safety profile.

Novel insights into the ecDNA formation mechanism involving MSH3 in methotrexate­resistant human colorectal cancer cells.

Extrachromosomal DNAs (ecDNAs), also known as double minutes (DMs), can induce a fast increase in gene copy numbers and promote the development of cancer, including drug resistance. MutS homolog 3 (MSH3), a key protein in mismatch repair, has been indicated to participate in the regulation of DNA double­strand break (DSB) repair, which has been reported to be associated with the formation of ecDNAs. However, it remains unclear whether MSH3 can influence drug resistance via ecDNAs in cancer. In the present study, high MSH3 expression was observed in methotrexate (MTX)­resistant HT29 cells [DM­ and homogeneously staining region (HSR)­containing cells] compared with parental HT29 cells. Additionally, decreased amounts of ecDNAs, HSRs and amplified genes locating on ecDNAs and HSRs were detected following depletion of MSH3 and this could be reversed by overexpressing MSH3 in DM­containing cells. No corresponding changes were found in HSR­containing cells. The present study further verified the involvement of MSH3­regulated DNA DSB repair pathways in the formation of ecDNAs by detecting the expression of core proteins and pathway activity. Furthermore, expulsion of ecDNAs/HSRs was detected and increased frequencies of micronuclei/nuclear buds with dihydrofolate reductase (DHFR) signals were observed in MSH3­depleted DM­containing cells. Finally, changes in MSH3 expression could affect DHFR amplification­derived DHFR expression and cell sensitivity to MTX, suggesting that MSH3 may influence cancer drug resistance by altering the amount of ecDNAs. In conclusion, the present study revealed a novel mechanism involving MSH3 in the regulation of ecDNAs by DSB repair, which will have clinical value in the treatment of ecDNA­based drug resistance in cancer.

Elevated expression of astrocyte elevated gene-1 (AEG-1) is correlated with cisplatin-based chemoresistance and shortened outcome in patients with stages III-IV serous ovarian carcinoma.

AIMS: To correlate astrocyte elevated gene-1 (AEG-1) expression with the clinicopathological features and outcome of patients with stages III-IV ovarian serous carcinoma, and to clinically assess the involvement of AEG-1 in acquired cisplatin resistance. METHODS AND RESULTS: The frequency and intensity of immunohistochemical AEG-1 expression increased in a step-wise fashion from normal to chemosensitive to chemoresistant tissues. These observations were confirmed by Western blot analysis. AEG-1 expression level was correlated with lymph nodal metastasis, histological differentiation, residual tumour size and response to primary chemotherapy. Five-year progression-free survival (PFS) and overall survival (OS) rates were lower in the high-expression group than that in the low-expression group. AEG-1 overexpression was an independent but poor prognostic factor in the OS and PFS of these patients, as determined by multivariate Cox regression analysis. Multivariate logistic regression analysis revealed that the presence of cisplatin-based chemoresistance was significantly associated with expression level of AEG-1 and the degree of residual disease (P = 0.0001 and P = 0.0027, respectively). CONCLUSION: Our findings indicate that tumour AEG-1 overexpression is associated with poor prognosis and cisplatin resistance in advanced serous ovarian cancer.

Methionine synthase reductase A66G polymorphism contributes to tumor susceptibility: evidence from 35 case-control studies.

Methionine synthase reductase (MTRR) gene is involved in tumorigenesis by regulating DNA methylation through activation of methionine synthase (MTR). MTRR is polymorphic at nucleotide 66 (A-to-G) and the resulting variant enzyme has a lower affinity for MTR. The reported associations of MTRR A66G polymorphism with cancer risk are contradictory. Therefore, we performed a meta-analysis to better assess the associations, including 18,661 cases and 27,678 controls from 35 studies. Crude ORs with 95% CIs were used to assess the strength of association between the MTRR A66G polymorphism and cancer risk. The pooled ORs were performed for homozygote model (GG vs. AA), heterozygote model (GG vs. GA), recessive genetic model (GG vs. GA + AA), and dominant genetic model (GG + GA vs. AA), respectively. Overall, results indicated that the G allele and GG variant genotypes were associated with a significantly increased cancer risk (G vs. A: OR, 1.039; 95% CI, 1.009-1.078; homozygote model: OR, 1.094; 95% CI, 1.006-1.191). In subgroup analysis by ethnicity, significant increased risks were found among Asians with G allele (G vs. A: OR, 1.063; 95% CI, 1.011-1.119; homozygote model: OR, 1.189; 95% CI, 1.055-1.341; recessive model: OR, 1.197; 95% CI, 1.068-1.341). For stratification analysis, the cancer types with fewer than three studies were categorized into "other cancers", and the results indicated that there was a significant elevated cancer risk in "other cancers" in all genetic models, not in colorectal cancer, lymphoid leukemia or breast cancer. In summary, our study suggests that the MTRR A66G polymorphism is a potential biomarker for cancer risk.

DJ-1 may contribute to metastasis of non-small cell lung cancer.

Lung cancer is a leading cause of cancer-related death, about 40% human non-small cell lung cancer (NSCLC) patients showed lymph node involvements. However, the precise mechanism for the metastasis is still not fully understood. This study was to analyze the potential molecular mechanism for lung cancer metastasis. In the current study, proteomics analysis by two-dimensional electrophoresis (2-DE) was performed first to identify the differentially expressed protein between the higher metastasis lung adenocarcinoma cell line Anip973 and the lower metastasis lung adenocarcinoma cell line AGZY83-a. We confirmed the result by RT-PCR, immunoblotting and immunocytochemistry analyses in these two cell lines. Then we examined the expression of the differentially expressed protein in tumor tissues of NSCLC patients by immunoblotting and immunohistochemistry analyses. Using 2-DE analysis, we have identified DJ-1 was expressed higher in the higher metastasis Anip973 compared to the parental cell line AGZY83-a, that was confirmed by RT-PCR, immunoblotting and immunocytochemistry analyses. In NSCLC patients' tumor tissues study, immunoblotting data showed that, DJ-1 expression level was significantly higher in 72.2% (13/18) of NSCLC tissue samples compared to that in paired normal lung tissues (P = 0.044). Immunohistochemistry analysis demonstrated increased DJ-1 expression in 85 NSCLC tumor tissue samples compared with 7 normal lung tissue samples (P = 0.044). DJ-1 expression was also found to be significantly correlated with cancer lymphatic metastasis (P = 0.039). DJ-1 might contribute to the metastasis of NSCLC.

A New Clue for the Late Eocene Freshwater Ecosystem of Central China Evidenced by New Fossils of Trapa L. and Hemitrapa Miki (Lythraceae).

Both Trapa L. and the extinct Hemitrapa Miki are aquatic plants in the family Lythraceae, with abundant fossil records in Eurasia and North America in the Cenozoic. However, documented materials are mainly based on fruit and pollen grains without reliable leaf fossils. Here, we report fossil leaves, fruit, and roots of Trapa and fruit of Hemitrapa from the late Eocene of Weinan, the Weihe Basin of central China. The fossil leaves are identified as a new species, Trapa natanifolia Z. C. Han et H. Jia sp. nov., which represents the earliest known record of a Trapa leaf fossil. It is remarkably similar to extant species of Trapa, mostly due to the unique inflated petiole structures found in both of them. While displaying prominent intergeneric differences, the incomplete fossil fruits are assigned to Trapa sp. indet. and Hemitrapa sp. indet. The former is the earliest fossil fruit record of Trapa, and the latter represents the earliest fossil record of Hemitrapa found in Asia. These new fossil discoveries suggest that the divergence of Trapa and Hemitrapa occurred at least by the late Eocene. It is believed that modern Trapa most likely originated in China. Furthermore, this unexpected aquatic plant fossil assemblage indicates that central China was warm and humid, with freshwater ponds or lakes, in the late Eocene and not as arid as previously thought.

Middle Miocene lotus (Nelumbonaceae, Nelumbo) from the Qaidam Basin, Northern Tibet Plateau.

The Neogene environment and paleovegetation of today's semi-arid and arid Central Asia remain elusive. Little is known about the effect of paleoclimatic change on the distribution and ecological response mechanisms of aquatic plants, especially on the Tibetan Plateau. Here, we report a new species of Nelumbo Adanson, including leaves, receptacles, and fruits, namely Nelumbo delinghaensis sp. nov., from the Upper Youshashan Formation of the upper Middle Miocene in the northern Qaidam Basin on the Tibetan Plateau. The new species comprises centrally peltate leaves with 12−15 actinodromous primary veins and a receptacle embedded with ca. 15−30 fruits, with an unlobed central disc. Megafossils of lotus from northwest China broaden the geographical and stratigraphic ranges of Nelumbo. Our findings suggest that a large freshwater lake body surrounded by temperate forests and grassland developed in the Qaidam Basin during the late Middle Miocene, in sharp contrast to the present desert vegetation. The climate used to be sufficiently warm and moist enough to support a forest-steppe ecosystem with abundant freshwater bodies.

ATP-binding cassette B1 gene polymorphisms, mRNA expression and chemosensitivity to paclitaxel in non-small cell lung cancer cells.

BACKGROUND AND OBJECTIVE: The adenosine triphosphate (ATP)-binding cassette, sub-family B, member 1 (ABCB1) gene encodes P-glycoprotein (Pgp), which plays an important role in drug disposition by limiting intracellular uptake of paclitaxel. ABCB1 gene polymorphisms may alter the expression and function of Pgp, thereby influencing the response to chemotherapy. A panel of 17 non-small cell lung cancer (NSCLC) cell lines was used to investigate whether alterations in the ABCB1 gene or its mRNA expression correlated with in vitro chemosensitivity to paclitaxel. METHODS: Polymorphisms in the ABCB1 gene were evaluated by direct sequencing. mRNA expression levels were assessed by quantitative real-time reverse transcription PCR. In vitro chemosensitivity to paclitaxel was expressed as half-maximal inhibitory concentration values, using a tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based colorimetric assay. RESULTS: The variant allele frequencies for four ABCB1 gene polymorphisms were 14.71% for 2677G>T/A, 32.35% for 2734T>C, 23.53% for 3396C>T and 76.47% for 3435C>T. There was a significant positive correlation between ABCB1 mRNA expression and half-maximal inhibitory concentration values for paclitaxel (r=0.5322, P=0.0279). None of the four ABCB1 gene polymorphisms were associated with paclitaxel chemosensitivity or ABCB1 mRNA expression in the 17 cell lines. CONCLUSIONS: These in vitro results suggest that high ABCB1 mRNA expression may be a predictive biomarker for poor chemosensitivity to paclitaxel. The panel of NSCLC cell lines may provide clues and indications for establishing clinically useful relationships between a given polymorphism or level of gene expression and chemosensitivity to an anti-cancer agent.

Transcription factor AP-2beta: a negative regulator of IRS-1 gene expression.

Down-regulation of insulin receptor substrate-1 (IRS-1) expression could modify the ability of IRS-1 to fulfill its functions. It has been proposed that the phosphorylation of IRS-1 on serine residues could promote its degradation. However, few studies have investigated the transcriptional regulation of IRS-1 in the pathogenesis of insulin resistance. Genotyping for genome-wide single nucleotide polymorphisms revealed that the transcription factor activating enhancer-binding protein-2beta (AP-2beta) is a novel candidate gene for conferring susceptibility to obesity and type 2 diabetes. AP-2beta is expressed in adipose tissue and its expression is increased during the maturation of adipocytes. Overexpression of AP-2beta leads to adipocyte hypertrophy, directly inhibits adiponectin expression, and enhanced the expression of inflammatory adipokines such as IL-6 and MCP-1. In this study, we found that overexpression of AP-2beta in 3T3-L1 adipocytes impaired the promoter activity of IRS-1, and subsequently decreased mRNA and protein expression. Electrophoretic mobility shift assays showed that AP-2beta bound specifically to the IRS-1 promoter region. Furthermore, site-directed mutagenesis of the AP-2 binding site located at -362 to -351, relative to the transcription start site, markedly decreased AP-2-induced suppression of IRS-1 promoter activity, whereas other putative AP-2 binding sites did not. Our results clearly showed that AP-2beta directly decreased IRS-1 expression by binding to its promoter. Based on these findings, we speculate that the AP-2beta transcriptional factor is a unique regulator of IRS-1 and a candidate gene for insulin resistance.

Reduced expression and novel splice variants of ING4 in human gastric adenocarcinoma.

ING4, a new member of the ING (inhibitor of growth) family of tumour suppressor genes, has been found to be deleted or down-regulated in gliomas, breast tumours, and head and neck squamous cell carcinomas. The goal of the present study was to investigate whether the expression and alternative splicing of ING4 transcripts are involved in the initiation and progression of stomach adenocarcinoma. ING4 mRNA and protein expression was examined in gastric adenocarcinoma tissues and human gastric adenocarcinoma cell lines by RT-PCR, real-time RT-PCR, tissue microarray immunohistochemistry, and western blot analysis. Alterations in ING4 transcripts were determined through sequence analysis of ING4 cDNA. Our data showed that ING4 mRNA and protein were dramatically reduced in stomach adenocarcinoma cell lines and tissues, and significantly less in female than in male patients. We also found that reduced ING4 mRNA expression correlated with the stage of the tumour. Interestingly, by sequence analysis, we discovered five novel aberrantly spliced variant forms of ING4_v1 and ING4_v2. These variants cause a codon frame-shift and, eventually, deletion of the NLS or PHD domain contributing to the mislocalization of p53 and/or HAT/HDAC complexes and, subsequently, altered gene expression in gastric adenocarcinoma. These results suggest that attenuated and aberrant ING4 expression may be involved in the initiation and progression of stomach adenocarcinoma.