Characterization of Novel Murine and Human PDAC Cell Models: Identifying the Role of Intestine Specific Homeobox Gene ISX in Hypoxia and Disease Progression.

Therapy failure and metastasis-associated mortality are stumbling blocks in the management of PDAC in patients. Failure of therapy is associated to intense hypoxic conditions of tumors. To develop effective therapies, a complete understanding of hypoxia-associated changes in genetic landscape of tumors during disease progression is needed. Because artificially immortalized cell lines do not rightly represent the disease progression, studying genetics of tumors in spontaneous models is warranted. In the current study, we generated a spectrum of spontaneous human (UM-PDC1; UM-PDC2) and murine (HI-PanL, HI-PancI, HI-PanM) models representing localized, invasive, and metastatic PDAC from a patient and transgenic mice (K-rasG12D/Pdxcre/Ink4a/p16-/). These spontaneous models grow vigorously under hypoxia and exhibit activated K-ras signaling, progressive loss of PTEN, and tumorigenicity in vivo. Whereas UM-PDC1 form localized tumors, the UM-PDC2 metastasize to lungs in mice. In an order of progression, these models exhibit genomic instability marked by gross chromosomal rearrangements, centrosome-number variations, Aurora-kinase/H2AX colocalization, loss of primary cilia, and α-tubulin acetylation. The RNA sequencing of hypoxic models followed by qRT-PCR validation and gene-set enrichment identified Intestine-Specific Homeobox factor (ISX)-driven molecular pathway as an indicator PDAC aggressivness. TCGA-PAAD clinical data analysis showed high ISX expression correlation to poor survival of PDAC patients, particularly women. The functional studies showed ISX as a regulator of i) invasiveness and migratory potential and ii) VEGF, MMP2, and NFκB activation in PDAC cells. We suggest that ISX is a potential druggable target and newly developed spontaneous cell models are valuable tools for studying mechanism and testing therapies for PDAC.

Regional Cerebral Blood Flow in Mania: Assessment Using 320-Slice Computed Tomography.

Objectives: While evidence that episodes of mania in bipolar I are associated with changes in bioenergetic and regional cerebral blood flow (rCBF) and cerebral blood flow velocity (rCBFV), both the regions and the extent of these changes have not yet been defined. Therefore, we determined the pattern of regional cerebral perfusion mania patients and using patients with major depressive disorder (MDD) as positive controls and healthy participants as negative controls. Methods: Twenty participants with mania, together with 22 MDD patients and 24 healthy volunteers, were recruited for this study. On all participants, Transcranial Doppler (TCD) was conducted to measure rCBFV parameters, 320-slice CT was conducted to measure rCBF in the different cerebral artery regions, and hematological parameters were assessed. ANOVA and Pearson's tests were used for the statistical analysis. Results: Our data indicated that rCBF in the medial temporal lobe and hippocampus, especially in the left medial temporal lobe and the right hippocampus, was increased in the mania group compared with the control and MDD groups (p < 0.01). In contrast, rCBF in the medial temporal lobe and hippocampus was decreased in the depression group (p < 0.01) compared with healthy controls. In addition, values of rCBFV in the bilateral internal carotid arteries (ICAs) and middle cerebral arteries (MCA) were increased in mania (p < 0.01) in comparison to the MDD group. Whole blood viscosity and hematocrit as well as red blood cell sedimentation rate remained unchanged in all group (p > 0.05). Conclusions: In mania, rCBF is increased in the medial temporal lobe and hippocampus, with a corresponding increase in rCBFV in the same regions.

Identification of a novel human estrogen receptor-α splice variant able to enhance malignant biological behaviors of breast cancer cells.

Since the early 1990s, multiple human estrogen receptor-α (hER-α) splice variants have been identified, of which the majority contain ≥1 deleted exon, and some are expressed as proteins with modified functions from the wild-type 66 kDa hER-α (ER-α66). In the present study, a novel hER-α splice variant, ER-α30, was identified and cloned from clinical breast cancer tissue. The ER-α30 sequence lacked a ligand-binding domain and a ligand-dependent transcriptional activation domain but retained the N-terminal transcriptional activation domain, the DNA-binding domain and a partial hinge domain, and possesses a unique 10-amino-acid domain. The expression of ER-α30 was associated with ER-α66-negative and progesterone receptor-negative breast cancer. The 30 kDa protein was expressed in stably transfected MDA-MB-231 cells, and the overexpression of ER-α30 in MDA-MB-231 cells enhanced malignant biological behaviors, including cellular proliferation, migration and invasion in vitro. The results of the present study indicated that ER-α30 might represent a potential biomarker for breast cancer.

There are only four basic modes of cell death, although there are many ad-hoc variants adapted to different situations.

There have been enough cell death modes delineated in the biomedical literature to befuddle all cell death researchers. Mulling over cell death from the viewpoints of the host tissue or organ and of the host animal, we construe that there should be only two physiological cell death modes, i.e. apoptosis and senescent death (SD), as well as two pathological modes, i.e. necrosis and stress-induced cell death (SICD). Other death modes described in the literature are ad-hoc variants or coalescences of some of these four basic ones in different physiological or pathological situations. SD, SICD and necrosis kill useful cells and will thus trigger regeneration, wound healing and probably also scar formation. SICD and necrosis will likely instigate inflammation as well. Apoptosis occurs as a mechanism to purge no-longer useful cells from a tissue via phagocytosis by cells with phagocytic ability that are collectively tagged by us as scavengers, including macrophages; therefore apoptosis is not followed by regeneration and inflammation. The answer for the question of "who dies" clearly differentiates apoptosis from SD, SICD and necrosis, despite other similarities and disparities among the four demise modes. Apoptosis cannot occur in cell lines in vitro, because cell lines are immortalized by reprogramming the death program of the parental cells, because in culture there lack scavengers and complex communications among different cell types, and because culture condition is a stress to the cells. Several issues of cell death that remain enigmatic to us are also described for peers to deliberate and debate.

Cervical Cancers Manifest a High Rate of Infection by a High-Risk Human Papilloma Virus Subtype but a Very Low Rate of Infection by a Low-Risk Subtype in the Guiyang District of China.

The prevalence of infection by different genotypes of human papillomavirus (HPV) varies among different geographic areas. We studied the prevalence of infection by 21 HPV genotypes in cervical tissue specimens from 4213 women in the Guiyang district, that is located in the southwest of China and is dominated by minor ethnicities of Chinese, and 2074 cases in our cohort had pathological diagnosis available. The overall infection rate was 36.98%. Most (72.08%) infectors were positive for only one HPV subtype, with the remaining being cases infected by two or more subtypes. Infections by the HPV subtypes 16, 52 and 58 were the most prevalent, having rates of 34.66%, 16.03%% and 15.53%, respectively. The most common cervical lesions in HPV infections were genital warts, cervical cancer (CC) and cervical intraepithelial neoplasia (CIN). Age and age at first sexual activity were independent risk factors for HPV infections that in turn cause certain cervical lesions. Intriguingly, while 94.90% of the CC patients were infected by oncogenically high-risk (HR) HPV subtypes, only 2.75% and 2.29% of these patients were infected by oncogenically low-risk (LR) subtypes or other-subtypes with their oncogenicity unclear. The rates of infection by LR-HPVs and other-HPVs were also low, being 4.63% and 6.76%, respectively, in the patients with CIN that is a precursor lesion of CC, lower than the 8.54% and 18.20%, respectively, in the women without a cervical lesion. Our data provides an important foundation for prevention, diagnosis and treatment of HPV infection in Guiyang district and suggests that development of vaccines for prevention and treatment of CC in this area should first target the HPV subtypes 16, 52 and 58, but not subtype 18 as for many other places. It deserves study whether infections by certain LR-HPVs and other-HPVs may serve as attenuated live vaccines for prevention of CC.

It Is Imperative to Establish a Pellucid Definition of Chimeric RNA and to Clear Up a Lot of Confusion in the Relevant Research.

There have been tens of thousands of RNAs deposited in different databases that contain sequences of two genes and are coined chimeric RNAs, or chimeras. However, "chimeric RNA" has never been lucidly defined, partly because "gene" itself is still ill-defined and because the means of production for many RNAs is unclear. Since the number of putative chimeras is soaring, it is imperative to establish a pellucid definition for it, in order to differentiate chimeras from regular RNAs. Otherwise, not only will chimeric RNA studies be misled but also characterization of fusion genes and unannotated genes will be hindered. We propose that only those RNAs that are formed by joining two RNA transcripts together without a fusion gene as a genomic basis should be regarded as authentic chimeras, whereas those RNAs transcribed as, and cis-spliced from, single transcripts should not be deemed as chimeras. Many RNAs containing sequences of two neighboring genes may be transcribed via a readthrough mechanism, and thus are actually RNAs of unannotated genes or RNA variants of known genes, but not chimeras. In today's chimeric RNA research, there are still several key flaws, technical constraints and understudied tasks, which are also described in this perspective essay.

Protein multiplicity can lead to misconduct in western blotting and misinterpretation of immunohistochemical staining results, creating much conflicting data.

Western blotting (WB) and immunohistochemical staining (IHC) are common techniques for determining tissue protein expression. Both techniques require a primary antibody specific for the protein in question. WB data is band(s) on a membrane while IHC result is a staining on a tissue section. Most human genes are known to produce multiple protein isoforms; in agreement with that, multiple bands are often found on the WB membrane. However, a common but unspoken practice in WB is to cut away the extra band(s) and present for publication only the band of interest, which implies to the readers that only one form of protein is expressed and thus the data interpretation is straightforward. Similarly, few IHC studies discuss whether the antibody used is isoform-specific and whether the positive staining is derived from only one isoform. Currently, there is no reliable technique to determine the isoform-specificity of an antibody, especially for IHC. Therefore, cutting away extra band(s) on the membrane usually is a form of misconduct in WB, and a positive staining in IHC only indicates the presence of protein product(s) of the to-be-interrogated gene, and not necessarily the presence of the isoform of interest. We suggest that data of WB and IHC involving only one antibody should not be published and that relevant reports should discuss whether there may be protein multiplicity and whether the antibody used is isoform-specific. Hopefully, techniques will soon emerge that allow determination of not only the presence of protein products of genes but also the isoforms expressed.

Establishment of a Simple and Quick Method for Detecting Extended-Spectrum ß-Lactamase (ESBL) Genes in Bacteria.

Extended-spectrum ß-lactamase (ESBL) genes that render bacteria resistant to antibiotics are commonly detected using phenotype testing, which is time consuming and not sufficiently accurate. To establish a better method, we used phenotype testing to identify ESBL-positive bacterial strains and conducted PCR to screen for TEM (named after the patient Temoneira who provided the first sample), sulfhydryl reagent variable (SHV), cefotaxime (CTX)-M-1, and CTX-M-9, the 4 most common ESBL types and subtypes. We then performed multiplex PCR with 1 primer containing a biotin and hybridized the PCR products with gene-specific probes that were coupled with microbeads and coated with a specific fluorescence. The hybrids were linked to streptavidin-R-phycoerythrins (SA-PEs) and run through a flow cytometer, which sorted the fluorescently dyed microbeads and quantified the PEs. The results from single PCR, multiplex PCR, and cytometry were consistent with each other. We used this method to test 169 clinical specimens that had been determined for phenotypes and found 154 positive for genotypes, including 30 of the 45 samples that were negative for phenotypes. The CTX-M genotype tests alone, counting both positive and negative cases, showed 99.41% (168/169) consistency with the ESBL phenotype test. Thus, we have established a multiplex-PCR system as a simple and quick method that is high throughput and accurate for detecting 4 common ESBL types and subtypes.

Two RNAs or DNAs May Artificially Fuse Together at a Short Homologous Sequence (SHS) during Reverse Transcription or Polymerase Chain Reactions, and Thus Reporting an SHS-Containing Chimeric RNA Requires Extra Caution.

Tens of thousands of chimeric RNAs have been reported. Most of them contain a short homologous sequence (SHS) at the joining site of the two partner genes but are not associated with a fusion gene. We hypothesize that many of these chimeras may be technical artifacts derived from SHS-caused mis-priming in reverse transcription (RT) or polymerase chain reactions (PCR). We cloned six chimeric complementary DNAs (cDNAs) formed by human mitochondrial (mt) 16S rRNA sequences at an SHS, which were similar to several expression sequence tags (ESTs).These chimeras, which could not be detected with cDNA protection assay, were likely formed because some regions of the 16S rRNA are reversely complementary to another region to form an SHS, which allows the downstream sequence to loop back and anneal at the SHS to prime the synthesis of its complementary strand, yielding a palindromic sequence that can form a hairpin-like structure.We identified a 16S rRNA that ended at the 4th nucleotide(nt) of the mt-tRNA-leu was dominant and thus should be the wild type. We also cloned a mouse Bcl2-Nek9 chimeric cDNA that contained a 5-nt unmatchable sequence between the two partners, contained two copies of the reverse primer in the same direction but did not contain the forward primer, making it unclear how this Bcl2-Nek9 was formed and amplified. Moreover, a cDNA was amplified because one primer has 4 nts matched to the template, suggesting that there may be many more artificial cDNAs than we have realized, because the nuclear and mt genomes have many more 4-nt than 5-nt or longer homologues. Altogether, the chimeric cDNAs we cloned are good examples suggesting that many cDNAs may be artifacts due to SHS-caused mis-priming and thus greater caution should be taken when new sequence is obtained from a technique involving DNA polymerization.

Cloning of Porcine Pituitary Tumor Transforming Gene 1 and Its Expression in Porcine Oocytes and Embryos.

The maternal-to-embryonic transition (MET) is a complex process that occurs during early mammalian embryogenesis and is characterized by activation of the zygotic genome, initiation of embryonic transcription, and replacement of maternal mRNA with embryonic mRNA. The objective of this study was to reveal the temporal expression and localization patterns of PTTG1 during early porcine embryonic development and to establish a relationship between PTTG1 and the MET. To achieve this goal, reverse transcription-polymerase chain reaction (RT-PCR) was performed to clone porcine PTTG1. Subsequently, germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, zygotes, 2-, 4-, and 8-cell-stage embryos, morulas, and blastocysts were produced in vitro and their gene expression was analyzed. The results revealed that the coding sequence of porcine PTTG1 is 609-bp in length and that it encodes a 202-aa polypeptide. Using qRT-PCR, PTTG1 mRNA expression was observed to be maintained at high levels in GV- and MII-stage oocytes. The transcript levels in oocytes were also significantly higher than those in embryos from the zygote to blastocyst stages. Immunohistochemical analyses revealed that porcine PTTG1 was primarily localized to the cytoplasm and partially localized to the nucleus. Furthermore, the PTTG1 protein levels in MII-stage oocytes and zygotes were significantly higher than those in embryos from the 2-cell to blastocyst stage. After fertilization, the level of this protein began to decrease gradually until the blastocyst stage. The results of our study suggest that porcine PTTG1 is a new candidate maternal effect gene (MEG) that may participate in the processes of oocyte maturation and zygotic genome activation during porcine embryogenesis.

Learning about the Importance of Mutation Prevention from Curable Cancers and Benign Tumors.

Some cancers can be cured by chemotherapy or radiotherapy, presumably because they are derived from those cell types that not only can die easily but also have already been equipped with mobility and adaptability, which would later allow the cancers to metastasize without the acquisition of additional mutations. From a viewpoint of biological dispersal, invasive and metastatic cells may, among other possibilities, have been initial losers in the competition for resources with other cancer cells in the same primary tumor and thus have had to look for new habitats in order to survive. If this is really the case, manipulation of their ecosystems, such as by slightly ameliorating their hardship, may prevent metastasis. Since new mutations may occur, especially during and after therapy, to drive progression of cancer cells to metastasis and therapy-resistance, preventing new mutations from occurring should be a key principle for the development of new anticancer drugs. Such new drugs should be able to kill cancer cells very quickly without leaving the surviving cells enough time to develop new mutations and select resistant or metastatic clones. This principle questions the traditional use and the future development of genotoxic drugs for cancer therapy.

Improvement of Natamycin Production by Cholesterol Oxidase Overexpression in Streptomyces gilvosporeus.

Natamycin is a widely used antifungal antibiotic. For natamycin biosynthesis, the gene pimE encodes cholesterol oxidase, which acts as a signalling protein. To confirm the positive effect of the gene pimE on natamycin biosynthesis, an additional copy of the gene pimE was inserted into the genome of Streptomyces gilvosporeus 712 under the control of the ermE* promoter (permE*) using intergeneric conjugation. Overexpression of the target protein engendered 72% and 81% increases in the natamycin production and cell productivity, respectively, compared with the control strain. Further improvement in the antibiotic production was achieved in a 1 L fermenter to 7.0 g/l, which was a 153% improvement after 120 h cultivation. Exconjugants highly expressing pimE and pimM were constructed to investigate the effects of both genes on the increase of natamycin production. However, the co-effect of pimE and pimM did not enhance the antibiotic production obviously, compared with the exconjugants highly expressing pimE only. These results suggest not only a new application of cholesterol oxidase but also a useful strategy to genetically engineer natamycin production.

Culture at a Higher Temperature Mildly Inhibits Cancer Cell Growth but Enhances Chemotherapeutic Effects by Inhibiting Cell-Cell Collaboration.

Acute febrile infections have historically been used to treat cancer. To explore the underlying mechanism, we studied chronic effects of fever on cancer cell growth and chemotherapeutic efficacy in cell culture. We found that culturing cancer cells at 39°C mildly inhibited cell growth by arresting the cells at the G1 phase of the cell cycle. When cells were seeded in culture dishes at a lower density, e.g. about 1000-2000 cells per 35-mm dish, the growth inhibition was much greater, manifested as many fewer cell colonies in the 39°C dishes, compared with the results at a higher density seeding, e.g. 20,000 cells per dish, suggesting that cell-cell collaboration as the Allee effect in cell culture is inhibited at 39°C. Withdrawal of cells from serum enhanced the G1 arrest at 39°C and, for some cell lines such as A549 lung cancer cells, serum replenishment failed to quickly drive the cells from the G1 into the S and G2-M phases. Therapeutic effects of several chemotherapeutic agents, including clove bud extracts, on several cancer cell lines were more potent at 39°C than at 37°C, especially when the cells were seeded at a low density. For some cell lines and some agents, this enhancement is long-lasting, i.e. continuing after the cessation of the treatment. Collectively these results suggest that hyperthermia may inhibit cancer cell growth by G1 arrest and by inhibition of cell-cell collaboration, and may enhance the efficacy of several chemotherapeutic agents, an effect which may persist beyond the termination of chemotherapy.

Weaknesses and Pitfalls of Using Mice and Rats in Cancer Chemoprevention Studies.

Many studies, using different chemical agents, have shown excellent cancer prevention efficacy in mice and rats. However, equivalent tests of cancer prevention in humans require decades of intake of the agents while the rodents' short lifespans cannot give us information of the long-term safety. Therefore, animals with a much longer lifespan should be used to bridge the lifespan gap between the rodents and humans. There are many transgenic mouse models of carcinogenesis available, in which DNA promoters are used to activate transgenes. One promoter may activate the transgene in multiple cell types while different promoters are activated at different ages of the mice. These spatial and temporal aspects of transgenes are often neglected and may be pitfalls or weaknesses in chemoprevention studies. The variation in the copy number of the transgene may widen data variation and requires use of more animals. Models of chemically-induced carcinogenesis do not have these transgene-related defects, but chemical carcinogens usually damage metabolic organs or tissues, thus affecting the metabolism of the chemopreventive agents. Moreover, many genetically edited and some chemically-induced carcinogenesis models produce tumors that exhibit cancerous histology but are not cancers because the tumor cells are still mortal, inducer-dependent, and unable to metastasize, and thus should be used with caution in chemoprevention studies. Lastly, since mice prefer an ambient temperature of 30-32°C, it should be debated whether future mouse studies should be performed at this temperature, but not at 21-23°C that cold-stresses the animals.

CD44 alternative splicing and hnRNP A1 expression are associated with the metastasis of breast cancer.

CD44 is a transmembrane receptor for hyaluronic acid. CD44 pre-mRNA contains 19 exons, 9 of which are alternatively spliced. Among the CD44 spliced variants, the v4-7 variant, one of the v6 exon-containing isoforms that contains variable exon 4, 5, 6 and 7, confers metastatic potential to non-metastatic cells. Splicing of CD44 and the function of CD44 isoforms are different in breast cancer cells. hnRNP A1 is a ubiquitously expressed protein with an inhibitory function in pre-mRNA splicing. We showed that CD44v6 isoform, which includes all of the v6-containing mRNA isoforms, had the highest expression level in non-metatatic breast cancer cells (MCF7) when compared to the level in metastatic breast cancer cells (MDA-MB-231) and normal breast cells (MCF10A). Furthermore we showed that hnRNP A1 knockdown regulated splicing of CD44 differently in breast cancer cells. We showed here that CD44 isoform expression is completely different in MDA-MB-231 cells than that in MCF7 and MCF10A cells, whereas MCF7 and MCF10A cells had a similar expression pattern of CD44 isoforms. RT-PCR analysis of CD44v6 showed that MCF7 and MCF10A cells predominantly expressed the c5v6v7v8v9v10c6 isoform. However, in addition to this isoform, MDA-MB-231 cells also expressed the c5v6v8v9v10c6 and c5v6c6 isoforms. We also found that knockdown of hnRNP A1 significantly reduced the expression of c5v6v7v8v9v10c6 and c5v6v8v9v10c6, and promoted the expression of c5v6c6. hnRNP A1 knockdown significantly induced cell death. In addition, hnRNP A1 knockdown induced a decrease in cell invasion in the MDA-MB-231 cells. Our results indicate that the knockdown of hnRNP A1 has a specific function on the splicing of CD44 in breast cancer cells.

Hypothesis: Artifacts, Including Spurious Chimeric RNAs with a Short Homologous Sequence, Caused by Consecutive Reverse Transcriptions and Endogenous Random Primers.

Recent RNA-sequencing technology and associated bioinformatics have led to identification of tens of thousands of putative human chimeric RNAs, i.e. RNAs containing sequences from two different genes, most of which are derived from neighboring genes on the same chromosome. In this essay, we redefine "two neighboring genes" as those producing individual transcripts, and point out two known mechanisms for chimeric RNA formation, i.e. transcription from a fusion gene or trans-splicing of two RNAs. By our definition, most putative RNA chimeras derived from canonically-defined neighboring genes may either be technical artifacts or be cis-splicing products of 5'- or 3'-extended RNA of either partner that is redefined herein as an unannotated gene, whereas trans-splicing events are rare in human cells. Therefore, most authentic chimeric RNAs result from fusion genes, about 1,000 of which have been identified hitherto. We propose a hypothesis of "consecutive reverse transcriptions (RTs)", i.e. another RT reaction following the previous one, for how most spurious chimeric RNAs, especially those containing a short homologous sequence, may be generated during RT, especially in RNA-sequencing wherein RNAs are fragmented. We also point out that RNA samples contain numerous RNA and DNA shreds that can serve as endogenous random primers for RT and ensuing polymerase chain reactions (PCR), creating artifacts in RT-PCR.

SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene.

The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ron△165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed.

Isoforms of wild type proteins often appear as low molecular weight bands on SDS-PAGE.

Immunoblotting, after polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE), is a technique commonly used to detect specific proteins. SDS-PAGE often results in the visualization of protein band(s) in addition to the one expected based on the theoretical molecular mass (TMM) of the protein of interest. To determine the likelihood of additional band(s) being nonspecific, we used liquid chromatography - mass spectrometry to identify proteins that were extracted from bands with the apparent molecular mass (MM) of 40 and 26 kD, originating from protein extracts derived from non-malignant HEK293 and cancerous MDA-MB231 (MB231) cells separated using SDS-PAGE. In total, approximately 57% and 21% of the MS/MS spectra were annotated as peptides in the two cell samples, respectively. Moreover, approximately 24% and 36.2% of the identified proteins from HEK293 and MB231 cells matched their TMMs. Of the identified proteins, 8% from HEK293 and 26% from MB231 had apparent MMs that were larger than predicted, and 67% from HEK293 and 37% from MB231 exhibited smaller MM values than predicted. These revelations suggest that interpretation of the positive bands of immunoblots should be conducted with caution. This study also shows that protein identification performed by mass spectrometry on bands excised from SDS-PAGE gels could make valuable contributions to the identification of cancer biomarkers, and to cancer-therapy studies.

The other side of the coin: the tumor-suppressive aspect of oncogenes and the oncogenic aspect of tumor-suppressive genes, such as those along the CCND-CDK4/6-RB axis.

Although cancer-regulatory genes are dichotomized to oncogenes and tumor-suppressor gene s, in reality they can be oncogenic in one situation but tumor-suppressive in another. This dual-function nature, which sometimes hampers our understanding of tumor biology, has several manifestations: (1) Most canonically defined genes have multiple mRNAs, regulatory RNAs, protein isoforms, and posttranslational modifications; (2) Genes may interact at different levels, such as by forming chimeric RNAs or by forming different protein complexes; (3) Increased levels of tumor-suppressive genes in normal cells drive proliferation of cancer progenitor cells in the same organ or tissue by imposing compensatory proliferation pressure, which presents the dual-function nature as a cell-cell interaction. All these manifestations of dual functions can find examples in the genes along the CCND-CDK4/6-RB axis. The dual-function nature also underlies the heterogeneity of cancer cells. Gene-targeting chemotherapies, including that targets CDK4, are effective to some cancer cells but in the meantime may promote growth or progression of some others in the same patient. Redefining "gene" by considering each mRNA, regulatory RNA, protein isoform, and posttranslational modification from the same genomic locus as a "gene" may help in better understanding tumor biology and better selecting targets for different sub-populations of cancer cells in individual patients for personalized therapy.

3' Splice site sequences of spinal muscular atrophy related SMN2 pre-mRNA include enhancers for nearby exons.

Spinal muscular atrophy (SMA) is a human genetic disease which occurs because of the deletion or mutation of SMN1 gene. SMN1 gene encodes the SMN protein which plays a key role in spliceosome assembly. Although human patients contain SMN2, a duplicate of SMN1, splicing of SMN2 produces predominantly exon 7 skipped isoform. In order to understand the functions of splice site sequences on exon 7 and 8, we analyzed the effects of conserved splice site sequences on exon 7 skipping of SMN2 and SMN1 pre-mRNA. We show here that conserved 5' splice site sequence of exon 7 promoted splicing of nearby exons and subsequently reduced splicing of distant exons. However, to our surprise, conserved 3' splice site sequence of exon 7 and 8 did not promote splicing of nearby exons. By contrast, the mutation inhibited splicing of nearby exons and subsequently promoted splicing of distant exons. Our study shows that 3' splice sites of exon 7 and 8 contain enhancer for their splice site selection, in addition to providing cleavage sites.