Forward-reverse mutation cycles in cancer cell lines under chemical treatments.

Spontaneous forward-reverse mutations were reported by us earlier in clinical samples from various types of cancers and in HeLa cells under normal culture conditions. To investigate the effects of chemical stimulations on such mutation cycles, the present study examined single nucleotide variations (SNVs) and copy number variations (CNVs) in HeLa and A549 cells exposed to wogonin-containing or acidic medium. In wogonin, both cell lines showed a mutation cycle during days 16-18. In acidic medium, both cell lines displayed multiple mutation cycles of different magnitudes. Genomic feature colocalization analysis suggests that CNVs tend to occur in expanded and unstable regions, and near promoters, histones, and non-coding transcription sites. Moreover, phenotypic variations in cell morphology occurred during the forward-reverse mutation cycles under both types of chemical treatments. In conclusion, chemical stresses imposed by wogonin or acidity promoted cyclic forward-reverse mutations in both HeLa and A549 cells to different extents.

Non-mitotic proliferation of malignant cancer cells revealed through live-cell imaging of primary and cell-line cultures.

INTRODUCTION: Anti-mitosis has been a key strategy of anti-cancer therapies, targeting at a fundamental property of cancer cells, their non-controllable proliferation due to overactive mitotic divisions. For improved anti-cancer therapies, it is important to find out whether cancer cells can proliferate independent of mitosis and become resistant to anti-mitotic agents. RESULTS: In this study, live-cell imaging was applied to both primary-cultures of tumor cells, and immortalized cancer cell lines, to detect aberrant proliferations. Cells isolated from various malignant tumors, such as Grade-III hemangiopericytoma, atypical meningioma, and metastatic brain tumor exhibit distinct cellular behaviors, including amoeboid sequestration, tailing, tunneling, nucleic DNA leakage, as well as prokaryote-like division such as binary fission and budding-shedding, which are collectively referred to and reported as 'non-mitotic proliferation' in this study. In contrast, benign tumors including Grade-I hemangiopericytoma and meningioma were not obvious in such behaviors. Moreover, when cultured in medium free of any anti-cancer drugs, cells from a recurrent Grade-III hemangiopericytoma that had been subjected to pre-operation adjuvant chemotherapy gradually shifted from non-mitotic proliferation to abnormal mitosis in the form of daughter number variation (DNV) and endomitosis, and eventually regular mitosis. Similarly, when treated with the anti-cancer drugs Epirubicin or Cisplatin, the cancer cell lines HeLa and A549 showed a shift from regular mitosis to abnormal mitosis, and further to non-mitosis as the dominant mode of proliferation with increasing drug concentrations. Upon removal of the drugs, the cells reversed back to regular mitosis with only minor occurrences of abnormal mitosis, accompanied by increased expression of the stem cell markers ALDH1, Sox, Oct4 and Nanog. CONCLUSIONS: The present study revealed that various types of malignant, but not benign, cancer cells exhibited cellular behaviors indicative of non-mitotic proliferation such as binary fission, which was typical of prokaryotic cell division, suggesting cell level atavism. Moreover, reversible transitions through the three modes of proliferation, i.e., mitosis, abnormal mitosis and non-mitosis, were observed when anticancer drug concentrations were grossly increased inducing non-mitosis or decreased favoring mitosis. Potential clinical significance of non-mitotic proliferation in cancer drug resistance and recurrence, and its relationship with cancer stem cells are worthy of further studies.

Copy number variation profile-based genomic typing of premenstrual dysphoric disorder in Chinese.

Premenstrual dysphoric disorder (PMDD) affects nearly 5% of women of reproductive age. Symptomatic heterogeneity, together with largely unknown genetics, has greatly hindered its effective treatment. In the present study, analysis of genomic sequencing-based copy number variations (CNVs) called from 100 kb white blood cell DNA sequence windows by means of semisupervized clustering led to the segregation of patient genomes into the D and V groups, which correlated with the depression and invasion clinical types, respectively, with 89.0% consistency. Application of diagnostic CNV features selected using the correlation-based machine learning method enabled the classification of the CNVs obtained into the D group, V group, total patient group, and control group with an average accuracy of 83.0%. The power of the diagnostic CNV features was 0.98 on average, suggesting that these CNV features could be used for the molecular diagnosis of the major clinical types of PMDD. This demonstrated concordance between the CNV profiles and clinical types of PMDD supported the validity of symptom-based diagnosis of PMDD for differentiating between its two major clinical types, as well as the predominantly genetic nature of PMDD with a host of overlaps between multiple susceptibility genes/pathways and the diagnostic CNV features as indicators of involvement in PMDD etiology.

Highly Recurrent Copy Number Variations in GABRB2 Associated With Schizophrenia and Premenstrual Dysphoric Disorder.

OBJECTIVE: Although single-nucleotide polymorphisms in GABRB2, the gene encoding for GABAA receptors ß2 subunit, have been associated with schizophrenia (SCZ), it is unknown whether there is any association of copy number variations (CNVs) in this gene with either SCZ or premenstrual dysphoric disorder (PMDD). METHODS: In this study, the occurrences of the recurrent CNVs esv2730987 in Intron 6 and nsv1177513 in Exon 11 of GABRB2 in Chinese and German SCZ, and Chinese PMDD patients were compared to controls of same ethnicity and gender by quantitative PCR (qPCR). RESULTS: The results demonstrated that copy-number-gains were enriched in both SCZ and PMDD patients with significant odds ratios (OR). For combined-gender SCZ patients versus controls, about two-fold increases were observed in both ethnic groups at both esv2730987 (OR = 2.15, p = 5.32E-4 in Chinese group; OR = 2.79, p = 8.84E-3 in German group) and nsv1177513 (OR = 3.29, p = 1.28E-11 in Chinese group; OR = 2.44, p = 6.17E-5 in German group). The most significant copy-number-gains were observed in Chinese females at nsv1177513 (OR = 3.41), and German females at esv2730987 (OR=3.96). Copy-number-gains were also enriched in Chinese PMDD patients versus controls at esv2730987 (OR = 10.53, p = 4.34E-26) and nsv1177513 (OR = 2.39, p = 3.19E-5). CONCLUSION: These findings established for the first time the association of highly recurrent CNVs with SCZ and PMDD, suggesting the presence of an overlapping genetic basis with shared biomarkers for these two common psychiatric disorders.

Genomic subtyping of liver cancers with prognostic application.

BACKGROUND: Cancer subtyping has mainly relied on pathological and molecular means. Massively parallel sequencing-enabled subtyping requires genomic markers to be developed based on global features rather than individual mutations for effective implementation. METHODS: In the present study, the whole genome sequences (WGS) of 110 liver cancers of Japanese patients published with different pathologies were analyzed with respect to their single nucleotide variations (SNVs) comprising both gain-of-heterozygosity (GOH) and loss-of-heterozygosity (LOH) mutations, the signatures of combined GOH and LOH mutations, along with recurrent copy number variations (CNVs). RESULTS: The results, obtained based on the WGS sequences as well as the Exome subset within the WGSs that covered ~ 2.0% of the WGS and the AluScan-subset within the WGSs that were amplifiable by Alu element-consensus primers and covered ~ 2.1% of the WGS, indicated that the WGS samples could be employed with the mutational parameters of SNV load, LOH%, the Signature α%, and survival-associated recurrent CNVs (srCNVs) as genomic markers for subtyping to stratify liver cancer patients prognostically into the long and short survival subgroups. The usage of the AluScan-subset data, which could be implemented with sub-micrograms of DNA samples and vastly reduced sequencing analysis task, outperformed the usage of WGS data when LOH% was employed as stratifying criterion. CONCLUSIONS: Thus genomic subtyping performed with novel genomic markers identified in this study was effective in predicting patient-survival duration, with cohorts of hepatocellular carcinomas alone and those including intrahepatic cholangiocarcinomas. Such relatively heterogeneity-insensitive genomic subtyping merits further studies with a broader spectrum of cancers.

Forward and reverse mutations in stages of cancer development.

BACKGROUND: Massive occurrences of interstitial loss of heterozygosity (LOH) likely resulting from gene conversions were found by us in different cancers as a type of single-nucleotide variations (SNVs), comparable in abundance to the commonly investigated gain of heterozygosity (GOH) type of SNVs, raising the question of the relationships between these two opposing types of cancer mutations. METHODS: In the present study, SNVs in 12 tetra sample and 17 trio sample sets from four cancer types along with copy number variations (CNVs) were analyzed by AluScan sequencing, comparing tumor with white blood cells as well as tissues vicinal to the tumor. Four published "nontumor"-tumor metastasis trios and 246 pan-cancer pairs analyzed by whole-genome sequencing (WGS) and 67 trios by whole-exome sequencing (WES) were also examined. RESULTS: Widespread GOHs enriched with CG-to-TG changes and associated with nearby CNVs and LOHs enriched with TG-to-CG changes were observed. Occurrences of GOH were 1.9-fold higher than LOH in "nontumor" tissues more than 2 cm away from the tumors, and a majority of these GOHs and LOHs were reversed in "paratumor" tissues within 2 cm of the tumors, forming forward-reverse mutation cycles where the revertant LOHs displayed strong lineage effects that pointed to a sequential instead of parallel development from "nontumor" to "paratumor" and onto tumor cells, which was also supported by the relative frequencies of 26 distinct classes of CNVs between these three types of cell populations. CONCLUSIONS: These findings suggest that developing cancer cells undergo sequential changes that enable the "nontumor" cells to acquire a wide range of forward mutations including ones that are essential for oncogenicity, followed by revertant mutations in the "paratumor" cells to avoid growth retardation by excessive mutation load. Such utilization of forward-reverse mutation cycles as an adaptive mechanism was also observed in cultured HeLa cells upon successive replatings. An understanding of forward-reverse mutation cycles in cancer development could provide a genomic basis for improved early diagnosis, staging, and treatment of cancers.

Coevolution Theory of the Genetic Code at Age Forty: Pathway to Translation and Synthetic Life.

The origins of the components of genetic coding are examined in the present study. Genetic information arose from replicator induction by metabolite in accordance with the metabolic expansion law. Messenger RNA and transfer RNA stemmed from a template for binding the aminoacyl-RNA synthetase ribozymes employed to synthesize peptide prosthetic groups on RNAs in the Peptidated RNA World. Coevolution of the genetic code with amino acid biosynthesis generated tRNA paralogs that identify a last universal common ancestor (LUCA) of extant life close to Methanopyrus, which in turn points to archaeal tRNA introns as the most primitive introns and the anticodon usage of Methanopyrus as an ancient mode of wobble. The prediction of the coevolution theory of the genetic code that the code should be a mutable code has led to the isolation of optional and mandatory synthetic life forms with altered protein alphabets.

GABRB2 Haplotype Association with Heroin Dependence in Chinese Population.

Substance dependence is a frequently observed comorbid disorder in schizophrenia, but little is known about genetic factors possibly shared between the two psychotic disorders. GABRB2, a schizophrenia candidate gene coding for GABAA receptor ß2 subunit, is examined for possible association with heroin dependence in Han Chinese population. Four single nucleotide polymorphisms (SNPs) in GABRB2, namely rs6556547 (S1), rs1816071 (S3), rs18016072 (S5), and rs187269 (S29), previously associated with schizophrenia, were examined for their association with heroin dependence. Two additional SNPs, rs10051667 (S31) and rs967771 (S32), previously associated with alcohol dependence and bipolar disorder respectively, were also analyzed. The six SNPs were genotyped by direct sequencing of PCR amplicons of target regions for 564 heroin dependent individuals and 498 controls of Han Chinese origin. Interestingly, it was found that recombination between the haplotypes of all-derived-allele (H1; OR = 1.00) and all-ancestral-allele (H2; OR = 0.74) at S5-S29 junction generated two recombinants H3 (OR = 8.51) and H4 (OR = 5.58), both conferring high susceptibility to heroin dependence. Additional recombination between H2 and H3 haplotypes at S1-S3 junction resulted in a risk-conferring haplotype H5 (OR = 1.94x109). In contrast, recombination between H1 and H2 haplotypes at S3-S5 junction rescued the risk-conferring effect of recombination at S5-S29 junction, giving rise to the protective haplotype H6 (OR = 0.68). Risk-conferring effects of S1-S3 and S5-S29 crossovers and protective effects of S3-S5 crossover were seen in both pure heroin dependent and multiple substance dependence subgroups. In conclusion, significant association was found with haplotypes of the S1-S29 segment in GABRB2 for heroin dependence in Han Chinese population. Local recombination was an important determining factor for switching haplotypes between risk-conferring and protective statuses. The present study provide evidence for the schizophrenia candidate gene GABRB2 to play a role in heroin dependence, but replication of these findings is required.

Massive interstitial copy-neutral loss-of-heterozygosity as evidence for cancer being a disease of the DNA-damage response.

BACKGROUND: The presence of loss-of-heterozygosity (LOH) mutations in cancer cell genomes is commonly encountered. Moreover, the occurrences of LOHs in tumor suppressor genes play important roles in oncogenesis. However, because the causative mechanisms underlying LOH mutations in cancer cells yet remain to be elucidated, enquiry into the nature of these mechanisms based on a comprehensive examination of the characteristics of LOHs in multiple types of cancers has become a necessity. METHODS: We performed next-generation sequencing on inter-Alu sequences of five different types of solid tumors and acute myeloid leukemias, employing the AluScan platform which entailed amplification of such sequences using multiple PCR primers based on the consensus sequences of Alu elements; as well as the whole genome sequences of a lung-to-liver metastatic cancer and a primary liver cancer. Paired-end sequencing reads were aligned to the reference human genome to identify major and minor alleles so that the partition of LOH products between homozygous-major vs. homozygous-minor alleles could be determined at single-base resolution. Strict filtering conditions were employed to avoid false positives. Measurements of LOH occurrences in copy number variation (CNV)-neutral regions were obtained through removal of CNV-associated LOHs. RESULTS: We found: (a) average occurrence of copy-neutral LOHs amounting to 6.9% of heterologous loci in the various cancers; (b) the mainly interstitial nature of the LOHs; and (c) preference for formation of homozygous-major over homozygous-minor, and transitional over transversional, LOHs. CONCLUSIONS: The characteristics of the cancer LOHs, observed in both AluScan and whole genome sequencings, point to the formation of LOHs through repair of double-strand breaks by interhomolog recombination, or gene conversion, as the consequence of a defective DNA-damage response, leading to a unified mechanism for generating the mutations required for oncogenesis as well as the progression of cancer cells.

Mutations enabling displacement of tryptophan by 4-fluorotryptophan as a canonical amino acid of the genetic code.

The 20 canonical amino acids of the genetic code have been invariant over 3 billion years of biological evolution. Although various aminoacyl-tRNA synthetases can charge their cognate tRNAs with amino acid analogs, there has been no known displacement of any canonical amino acid from the code. Experimental departure from this universal protein alphabet comprising the canonical amino acids was first achieved in the mutants of the Bacillus subtilis QB928 strain, which after serial selection and mutagenesis led to the HR23 strain that could use 4-fluorotryptophan (4FTrp) but not canonical tryptophan (Trp) for propagation. To gain insight into this displacement of Trp from the genetic code by 4FTrp, genome sequencing was performed on LC33 (a precursor strain of HR23), HR23, and TR7 (a revertant of HR23 that regained the capacity to propagate on Trp). Compared with QB928, the negative regulator mtrB of Trp transport was found to be knocked out in LC33, HR23, and TR7, and sigma factor sigB was mutated in HR23 and TR7. Moreover, rpoBC encoding RNA polymerase subunits were mutated in three independent isolates of TR7 relative to HR23. Increased expression of sigB was also observed in HR23 and in TR7 growing under 4FTrp. These findings indicated that stabilization of the genetic code can be provided by just a small number of analog-sensitive proteins, forming an oligogenic barrier that safeguards the canonical amino acids throughout biological evolution.

A rapid fluorescence polarization-based method for genotypic detection of drug resistance in Mycobacterium tuberculosis.

Rapid detection of drug-resistant Mycobacterium tuberculosis is critical to the effective early treatment and prevention of the transmission of tuberculosis. However, conventional drug susceptibility tests for M. tuberculosis require up to several weeks. In the present study, the One Label Extension genotyping method was adapted for rapid detection of drug resistance-associated sequence variations in six genes of M. tuberculosis, viz. rpoB, rpsL, rrs, embB, katG, or inhA. The method utilizes polymerase chain reaction amplified fragments of the drug resistant genes as reaction templates, and proceeds with template-directed primer extension incorporating a fluorescence-labeled nucleotide, which is then measured by fluorescence polarization. A total of 121 M. tuberculosis isolates from clinical sputum specimens were examined by this genotyping method and verified by direct sequencing of polymerase chain reaction amplicons harboring previously reported mutational sites associated with M. tuberculosis drug resistance. Based on phenotyping results obtained from microbiology-based drug susceptibility tests, the sensitivity, specificity, and test efficiency estimated for One Label Extension assays were respectively 83.9 %, 95.5 %, and 92.4 % with ropB in rifampin resistance, 67.3 %, 97.1 %, and 84.3 % with rpsL and rrs in streptomycin resistance, 60.0 %, 96.0 %, and 91.4 % with embB in ethambutol resistance, 68.4 %, 94.9 %, and 86.3 % with inhA and katG in isoniazid resistance, and 74.1 %, 98.9 %, and 93.2 % in multiple drug resistance defined as resistance to at least both isoniazid and rifampin. In conclusion, examination of clinical sputum specimens by One Label Extension based genotyping provides a valid method for the rapid molecular detection of drug-resistant M. tuberculosis.

Copy number variation analysis based on AluScan sequences.

BACKGROUND: AluScan combines inter-Alu PCR using multiple Alu-based primers with opposite orientations and next-generation sequencing to capture a huge number of Alu-proximal genomic sequences for investigation. Its requirement of only sub-microgram quantities of DNA facilitates the examination of large numbers of samples. However, the special features of AluScan data rendered difficult the calling of copy number variation (CNV) directly using the calling algorithms designed for whole genome sequencing (WGS) or exome sequencing. RESULTS: In this study, an AluScanCNV package has been assembled for efficient CNV calling from AluScan sequencing data employing a Geary-Hinkley transformation (GHT) of read-depth ratios between either paired test-control samples, or between test samples and a reference template constructed from reference samples, to call the localized CNVs, followed by use of a GISTIC-like algorithm to identify recurrent CNVs and circular binary segmentation (CBS) to reveal large extended CNVs. To evaluate the utility of CNVs called from AluScan data, the AluScans from 23 non-cancer and 38 cancer genomes were analyzed in this study. The glioma samples analyzed yielded the familiar extended copy-number losses on chromosomes 1p and 9. Also, the recurrent somatic CNVs identified from liver cancer samples were similar to those reported for liver cancer WGS with respect to a striking enrichment of copy-number gains in chromosomes 1q and 8q. When localized or recurrent CNV-features capable of distinguishing between liver and non-liver cancer samples were selected by correlation-based machine learning, a highly accurate separation of the liver and non-liver cancer classes was attained. CONCLUSIONS: The results obtained from non-cancer and cancerous tissues indicated that the AluScanCNV package can be employed to call localized, recurrent and extended CNVs from AluScan sequences. Moreover, both the localized and recurrent CNVs identified by this method could be subjected to machine-learning selection to yield distinguishing CNV-features that were capable of separating between liver cancers and other types of cancers. Since the method is applicable to any human DNA sample with or without the availability of a paired control, it can also be employed to analyze the constitutional CNVs of individuals.

Effects of flavone 6-substitutions on GABAA receptors efficacy.

Flavones have been studied for their activities via benzodiazepine site on the type-A γ-aminobutyric acid (GABA(A)) receptors, for which knowledge on structure-efficacy relationships has been rather limited in comparison to that on structure-affinity relationships. The present study focused on flavone 6-substitution, implied in previous studies being relevant to efficacy. Structure analogs, each varying only at position 6, were compared, including 6-fluoroflavone, 6-chloroflavone, 6-bromoflavone, and 2'-hydroxyflavone analyzed in the present study, as well as 6,2'-dihydroxyflavone reported earlier. Radio-ligand binding assays, whole-cell patch-clamp, and mouse behavioral experiments were performed. In consistent with a previous report, the present whole-cell patch-clamp and animal behavior experiments demonstrated 6-bromoflavone to be a positive modulator at GABA(A) receptors acting through flumazenil-sensitive high-affinity benzodiazepine site. In contrast, the other two 6-haloflavones were both neutralizing modulators. In vitro electrophysiological and in vivo animal experiments showed that 2'-hydroxyflavone was a neutralizing modulator, different in efficacy from its structural analog, 6,2'-dihydroxyflavone, a negative modulator of GABA(A) receptors. The fact that flavone analogs differing only at position 6 showed drastically different pharmacological properties clearly points to 6-substitution being an important determinant of efficacy. The results suggest that a large width of the first atom on the 6-substituent favors a high binding affinity of the 6-substituted flavone, whereas a large overall volume of the 6-substituent favors positive modulator activity, which could be modified by, e.g., 2'-hydroxyl substitution. These findings have contributed to the understanding of quantitative structure-efficacy relationships for flavones acting at GABA(A) receptors, and hence facilitation of flavone-based drug development.

Genetic code mutations: the breaking of a three billion year invariance.

The genetic code has been unchanging for some three billion years in its canonical ensemble of encoded amino acids, as indicated by the universal adoption of this ensemble by all known organisms. Code mutations beginning with the encoding of 4-fluoro-Trp by Bacillus subtilis, initially replacing and eventually displacing Trp from the ensemble, first revealed the intrinsic mutability of the code. This has since been confirmed by a spectrum of other experimental code alterations in both prokaryotes and eukaryotes. To shed light on the experimental conversion of a rigidly invariant code to a mutating code, the present study examined code mutations determining the propagation of Bacillus subtilis on Trp and 4-, 5- and 6-fluoro-tryptophans. The results obtained with the mutants with respect to cross-inhibitions between the different indole amino acids, and the growth effects of individual nutrient withdrawals rendering essential their biosynthetic pathways, suggested that oligogenic barriers comprising sensitive proteins which malfunction with amino acid analogues provide effective mechanisms for preserving the invariance of the code through immemorial time, and mutations of these barriers open up the code to continuous change.

The genomics of LUCA.

To understand the nature and evolution of LUCA, or Last Universal Common Ancestor, the minimum genome of LUCA has been identified based on the genes common to the eight primitive Euryarchaea and Crenarchaea species Methanopyrus kandleri, Methanothermobacter thermautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Aeropyrum pernix and Pyrobaculum aerophilum, together with the methanogenesis genes of the primitive methanogens. The 424 protein encoding genes in the minimum LUCA genome exceed significantly the 150-340 genes estimated to be present in a minimal proteome compatible with life. Thus LUCA was not a minimal organism but the first modern organism equipped with a DNA genome and the universal genetic code. The hyperthermophilic, Methanopyrus-proximal LUCA is consistent with a Hot Cross Origin of life which proposes that early heterotrophic life forms in the cooler temperature zones invented methanogenesis and a DNA genome upon their adaptation to the hydrothermal vents, where life flourished massively on lithoautotrophy supported by carbon dioxide and hydrogen, thereby leading to the rise of LUCA.

Polyphasic evidence delineating the root of life and roots of biological domains.

Twenty different lines of polyphasic evidence obtained from tRNA and protein sequences, anticodon usages, gene contents, metabolism and geochemistry have made possible the identification of a Last Universal Common Ancestor (LUCA) phylogenetically located proximal to the hyperthermophilic methanogenic archaeon Methanopyrus. Combined with analysis of high-similarity cross-domain tRNA pairs, the evidence also suggests a Thermotoga-proximal Last Bacterial Common Ancestor (LBACA) that originated from Crenarchaeota close to Aeropyrum, and a Plasmodium-proximal Last Eukaryotic Common Ancestor (LECA) derived from Ferroplasma through endosymbiosis.