Molecular mechanism of stomach carcinogenesis

Gene changes in multiple oncogenes, multiple growth factors and multiple tumor-suppressor genes are observed in stomach cancer. Among them, those most commonly implicated in both well-differentiated adenocarcinoma and poorly differentiated adenocarcinoma are inactivation (mutations and allele loss) of the p53 gene, and activation (abnormal expression and amplification) of the c-met gene. Moreover, they occur at an early stage of stomach carcinogenesis. In addition, loss of heterozygosity (LOH) on chromosome 5q (APC locus) is frequently associated with well-differentiated adenocarcinoma. LOH on chromosome 18q (DCC locus) and LOH of thebcl-2 gene also are common events of well-differentiated adenocarcinoma. LOH on chromosomes 1q and 7q may be involved in the progression of well-differentiated adenocarcinoma. Conversely, the development of poorly differentiated adenocarcinoma, in addition to changes in p53 and c-met genes, requires reduction or dysfunction of cadherin. Overexpression ofbcl-2 protein is observed in poorly differentiated adenocarcinoma or signetring cell carcinoma. Moreover, the K-sam gene is amplified preferentially in poorly differentiated adenocarcinoma or scirrhous carcinoma. K-sam amplification in scirrhous carcinoma often occurs independently of c-met gene amplification. LOH on chromosome 1p also is relatively common in poorly differentiated adenocarcinoma. Exceptionally, signetring cell carcinoma shares APC mutations. There are some differences in expression of the growth-factor/receptor system between well-differentiated adenocarcinoma and poorly differentiated adenocarcinoma. Moreover, interaction between cell-adhesion molecules in tumor cells expressing c-met and hepatocyte growth factor (HGF) from stromal cells is linked with morphogenesis of two histological types of stomach cancer. Intestinal metaplasia and adenoma of the stomach also contain p53 mutation and K-ras mutations ortpr-met rearrangement. Taken together, different genetic pathways of stomach carcinogenesis may exist for poorly differentiated and well-differentiated stomach cancers. Some of the latter may develop by a cumulative series of gene alterations similar to those of colorectal cancer.Abbreviation LOH loss of heterozygosity The Journal of Cancer Research and Clinical Oncology publishes in loose succession Editorials and Guest editorials on current and/or controversial problems in experimental and clinical oncology. These contributions represent exclusively the personal opinion of the author The Editors     

Effects of retinoic acid on NIH3T3 cell transformation by the H-ras oncogene

Summary Exposure of NIH3T3 cells to retinoic acid resulted in a dose-dependent modulation of transformed focus formation after transfection with an activated H-ras oncogene. Inhibition induced by 10 M retinoic acid was maximal at 21.4% of control values. Maximal inhibition of transformation was found after exposure to 10 M retinoic acid between days 0 and 3 of the transfection period. This concentration was also inhibitory for colony formation upon transfection of the non-transforming geneaph, suggesting that retinoic acid acts primarily on the process of transfection to inhibit focus or colony formation. Exposure to retinoic acid during the late period of the transfection protocol (days 14–20) resulted in alterations in focus morphology. A transformed cell line containing H-ras underwent reversion of the transformed phenotype after 4 weeks of treatment with retinoic acid, as determined by alterations in cell morphology and anchorage-independent growth. Phenotypic reversion was not associated with changes in the expression of the exogenous H-ras or endogenous c-myc or c-fos oncogenes.Abbreviations DMEM Dulbecco's modified Eagle medium - SSC saline sodium citrate - TGF transforming growth factor This investigation was supported by grants CA 52925, CA 13343 and ES 00260     

“Atypical” multidrug resistance in human ovarian cancer cell line A2780 selected for resistance to doxorubicin (A2780 DX3)

Human ovarian cancer cells A2780, selected for resistance to doxorubicin (A2780-DX3), are crossresistant to various other topoisomerase-II-targeted drugs but not to vinblastine. The parental cell line was very sensitive to doxorubicin-, mitoxantrone- or etoposide (VP16)-induced DNA single-strand breaks, under deproteinizing conditions. In contrast, little or no DNA strand breakage was seen in resistant A2780-DX3 cells, even at very high concentrations, indicating a good correlation, with cytotoxicity. No significant alterations in cellular drug uptake were observed in DX3 cells. Further studies showed that the nuclei isolated from resistant cells were also resistant to mitoxantroneor VP16-induced single-strand breaks, indicating that nuclear modifications in resistant cells are responsible for this resistance. Catalytic activity in crude nuclear extracts from wild-type and DX3 cells was almost equal. However, an assay that specifically measures generation of 5-protein-linked breaks in³²P-labeled 3 DNA revealed that, DNA cleavage activity in nuclear extract from the DX3 cell line is profoundly resistant to a stimulation by VP16. These data indicate that stimulation of topoisomerase-II-mediated DNA cleavage is responsible for topoisomerase-II-targeted drugcytotoxicity rather than loss of normal topoisomerase catalytic function. These data support the hypothesis that A2780-DX3 cells display an atypical multidrug resistance.Abbreviations MDR multidrug resistance - SSB Single-strand break     

In vitro-induced resistance to the deoxycytidine analogues cytarabine (AraC) and 5-aza-2′-deoxycytidine (DAC) in a rat model for acute myeloid leukemia is mediated by mutations in the deoxycytidine kinase (dck) gene

The deoxycytidine kinase (dck) gene encodes the enzyme responsible for the metabolic activation of the antileukemic drugs cytosine arabinoside (AraC) and 5-aza-2-deoxycytidine (decitabine, DAC). Thedck locus was analyzed at the chromosomal and the molecular level in a model of rat leukemic cell lines, in which AraC and DAC resistance was induced, that was marked bydck deficiency. At the chromosomal level, karyotype analysis of metaphase spreads revealed the presence of an aberrant 2q+ chromosome in the AraC-resistant cell line and a (Xq;11q) translocation in its subclone RA/7. The DAC-resistant lines were identical to the parental RCL/O. Fluorescence in situ hybridization on normal rat fibroblast metaphase spreads localized the ratdck gene to chromosome 14q21-q22, a region that was not involved in any of the observed karyotypic aberrations. Analysis at the molecular level revealed an identical rearrangement of thedck gene in the AraC-resistant cell line RCL/A and its subclone RA/7 that resulted in the absence ofdck expression, as assessed by RT-PCR. No genomic rearrangements were observed in a DAC-resistant cell line RCL/D or in its subclone RD/1. However, detection of a single-stranded conformation polymorphism (SSCP) allowed the identification of a single C to G substitution (His to Gln) in thedck cDNA of the DAC-resistant RD/1 clone. The data demonstrate that exposure to AraC and DAC induces a resistant phenotype marked by functionaldck deficiency that may be the consequence of mutations occurring in thedck gene.This work was supported by grant KWF 90-02 from the Dutch Cancer Foundation     

Expression of myc and ras oncogenes in two newly established neuroblastoma cell lines

Summary Two new neuroblastoma cell lines, KG-MH and KM-YH have been established from fresh tumour samples. In vitro growth characteristics are presented together with a karyological analysis. Northern and Southern blot experiments have been performed using molecularly cloned probes for c-myc, N-myc, c-Ha-ras, c-Ki-ras, and N-ras oncogenes. Both cell lines showed expression for N-myc, while c-myc expression was not detected. Cell line KM-YH, with a rather long population doubling time of 78 h, showed additional expression for the three ras genes.     

Amplification of the thymidylate synthase gene in anN 10-propargyl-5,8-dideazafolic-acid-resistant human leukemia,MOLT-3 cell line developed in pteroylglutamic acid,but not in leucovorin

The types of folates used during the development of resistance to methotrexate have been suggested to play an important role in the mechanisms of established resistance. In this study, effects of reduced and oxidized folates on the development of resistance to a thymidylate synthase (TS) inhibitor,N ¹⁰-propargyl-5,8-dideazafolic acid (CB3717), were exmined in the human leukemia cell line MOLT-3. MOLT-3 cells were made resistant to CB3717 by soft-agar cloning in RPMI-1640 medium with either pteroylglutamic acid (PGA) or a more physiological folate (10 nM leucovorin). A 40-fold CB3717-resistant subline developed in PGA (MOLT-3/CB3717₄₀-PGA) showed amplification of the TS gene with a concomitant increased level in the gene expression. A 200-fold CB3717-resistant subline (MOLT-3/CB3717₂₀₀-PGA), which was derived from MOLT-3/CB3717₄₀-PGA, showed further enhancement of amplification of the TS gene. In contrast, even a 200-fold CB3717-resistant subline developed in leucovorin (MOLT-3/CB3717₂₀₀-LV) showed neither amplification nor overexpression of the TS gene. Both MOLT-3/CB3717₂₀₀-PGA and MOLT-3/CB3717₂₀₀-LV cells showed decreased membrane transport of PGA as well as methotrexate. These results suggest that the types of folates used during the development of CB3717 resistance may play a role in resistance, and that impaired transport of PGA, in CB3717-resistant MOLT-3 cells developed in PGA, might have accelerated amplification of the TS gene.Abbreviations CB3717 N ¹⁰-propargyl-5,8-dideazafolic acid - DHFR dihydrofolate reductase - FCS fetal calf serum - IC ₅₀ 50% inhibitory concentration for cell growth by MTT assay - LV leucovorin, 5-formyltet-rahydrofolate - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) - PGA pteroylglutamic acid - TS thymidylate synthase     

Adrenergic agents,but not triiodo-L-thyronine induce c-fos and c-myc expression in the rat heart

Summary We have examined the expression of two nuclear-acting oncogenes, c-fos and c-myc in the rat heart following administration of hormones implicated in the development of cardiac hypertrophy. A single injection of norepinephrine (2.5 g/kg to 2.5 mg/kg) led to transient increases in the levels of both c-fos and c-myc mRNA. The response was sequential: elevated levels of c-fos mRNA were first observed 15 min after treatment and peaked at 1 h whilst c-myc mRNA levels increased 30 min after treatment and peaked at 2h. The response of both cellular oncogenes to norepinephrine was reduced significantly by a blockade but (3 blockade was less effective. Administration of triiodo-L-thyronine (0.25 mg/kg), a level known to promote cardiac hypertrophy, did not produce elevated levels of c-fos or c-myc mRNA. In an initial study, it was possible to demonstrate induction of c-fos and c-myc in rat hearts perfused in vitro with medium containing 2x10^–7 M norepinephrine. These results provide support for the notion that c-fos and c-myc expression may play a transducing role in the development of adrenergic-mediated, but not thyroid hormone-mediated cardiac hypertrophy.     

Carbohydrate-dependent binding of human myeloid leukemia cell lines to neoglycoenzymes,matrix-immobilized neoglycoproteins,and bone marrow stromal cell layers

Summary The presence of sugar receptors on human myeloid leukemia cells was comparatively assessed by a highly sensitive binding assay, employing a panel of 14 types of neoglycoenzymes (chemically glycosylatedEscherichia coli -galactosidase). The selected carbohydrate ligands mainly encompass common components of natural glycoconjugates as mono- or disaccharides. The monocytoid cells of the THP-1 line, the very young myeloblasts and the myeloblasts of the lines KG-1a and KG-1, the promyelocytes of the HL-60 line, and the early myeloblasts/erythroblasts of the K-562 line displayed a nonuniform pattern of specific binding with quantitative differences at a fixed, nonsaturating concentration of the probes. Scatchard analysis in four cases corroborated the indication of cell-type-related differences between the various cell lines. To test whether the detectable cellular sugar-binding sites can mediate adhesion to glycoligands, a rather simple model matrix of nitrocellulose-immobilized neoglycoproteins was first used. In comparison to the carbohydrate-free carrier protein significant cell adhesion was observed primarily with neoglycoproteins that exposed galactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, and fucose moieties among the 11 tested types of carbohydrate residue. Subsequently, human bone marrow stromal cell layers were tested as a model matrix with increased levels of physiological relevance and complexity. Mixtures of carbohydrate and neoglycoprotein were employed as inhibitors of an interaction via lectins between the stromal and the tumor cells. The carbohydrate-dependent alterations of this parameter revealed cell-type-associated properties. Tumor cell binding was significantly decreased for not more than two lines with the effective sugars, namelyN-acetylgalactosamine, mannose, fucose, and sialic acid.     

Association of genetic alterations of c-myc,c-fos,and c-Ha-ras proto-oncogenes in colorectal tumors

Using Northern and dot-blot analysis we examined normal and tumor tissue from 29 patients with colorectal carcinomas for the expression and amplification of c- myc,c-fos and c-Ha-ras proto-oncogenes. Overexpression of c-myc (6/24), c-fos (4/24), and c-Ha- ras (9/23) was found. For the c- fos proto-oncogene we also have observed decreased levels of expression in 13 percent (3/24) of the cases analyzed. Gene amplification appeared to be a rare event in these tumors and was found in 3/29 (10 percent) tumors for c- myc and in 1/29 (3 percent) for c- fos protooncogene.Curves for overall survival and for disease-free survival failed to show a significant tendency in these parameters to be poorer in tumors with alterations of gene expression for any of the proto-oncogenes analyzed. Despite the biologic importance of these genetic alterations in the etiology of colorectal tumors, levels of c- myc,c- fos,and c-Ha- ras gene expression separately or together cannot be considered as prognostic factors for clinical outcome of the disease.This work was supported by grant 507750/87 of the Conselho Nacional de Desenvolvimento Cientificoe Tecnologico.     

Flowcytometric analysis of multidrug-resistance-associated antigen (P-Glycoprotein) and DNA ploidy in human colon cancer

Summary In many cell systems, resistance to cytotoxic drugs is acquired by the amplification and/or overexpression of the multidrug resistance (mdr) gene, which codes for the glycoprotein, p170 (P-glycoprotein). Moreover, in a variety of malignant tumours there is increasing evidence of the relationship between the DNA ploidy pattern of patients and their prognosis. In this study we aimed to evaluate these two potential indicators of constitutive drug resistance in human colorectal tumours. We employed a method to quantify simultaneously, on a per cell basis,mdr gene expression (using the C219 monoclonal antibody for P-glycoprotein) and nuclear DNA content with high-resolution bivariate flow cytometry. The study was performed on a human coloncarcinoma-derived cell line (LoVo) and its doxorubicinresistant variant (LoVo/Dx) and on tumour samples and adjacent normal mucosa from 35 untreated patients with colon cancer. The P-glycoprotein was found in both LoVo and LoVo/Dx cells with levels slightly lower in the parental than in the resistant subline (P, NS). A multidrug-resistant specific probe for mRNA expression and Western blot assay confirmed the specificity of p170 expression. All of the colon cancer with unimodal diploid DNA distribution and all the normal colonic mucosa samples showed P-glycoprotein expression, without a statistically significant difference in median values between tumours and normal samples. Tumours with bimodal DNA distribution showed median values of P-glycoprotein expression of their hyperdiploid cell clones significantly higher than those of their diploid clones and of the tumours with unimodal DNA distribution (P<0.005). Our results show the feasibility of bivariate flow-cytometric analysis of P-glycoprotein expression and DNA content on clinical material and support the hypothesis that the MDR phenotype and DNA ploidy together may influence the biological behaviour of colon cancer in vivo.Abbreviations MDR multidrug resistance phenotype - mdr multidrug resistance gene - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline Research supported in part by C.N.R. target projects Oncology and Biotechnology and Bioinstrumentation, by A.I.R.C. and by I.R.C.C.S. San Matteo     

A human Burkitt’s lymphoma cell line carrying t(8;22) and t(14;18) translocations

A combination of chromosomal translocations associated with bcl-2 re-arrangement (t(14;18)) and c-myc re-arrangement (t(8;14), t(8;22), or t(2;8)) is a rare event. We describe the first cell line exhibiting t(14;18) and t(8;22), which will enable us to study the interactions of bcl-2 and c-myc systematically. Cell culture was started with circulating lymphoma cells from the peripheral blood of an adult male Caucasian patient with Burkitt’s lymphoma after the second relapse. The cells grew spontaneously without cytokines, fulfilled all criteria of a cell line and were analysed. An Epstein–Barr virus (EBV) genome-negative cell line (DoGKiT) has been established. RC-banding analysis of the chromosomes showed a complex karyotype with a modal number of 48, XY, dup(1)(q31;q44), t(8;22)(q24;q11), der(10), t(14;18)(q32;q21), add(16)(pter), dup(17)(q12q24), +der(18), +20. The combination of t(8;22)(q24;q11), a variant translocation of Burkitt’s lymphoma and t(14;18)(q32;q21), typical for follicular lymphoma (FL), was confirmed by FISH and SKY-analysis. Surface marker studies of the cell line showed that the cells were positive for CALLA (CD10), CD19, cyCD22, cyCD79a and HLA-DR and negative for TdT, IgM, CD5 and CD23. To our knowledge, this is the first established cell line carrying these two translocations. In contrast to already established cell lines carrying the more common combination of t(8;14)(q24;q32) and t(14;18)(q32;q21) with both IgH alleles being involved in translocations, the cell line DoGKiT carries only one translocated IgH allele. This cell line may serve as an important tool in the study of the combination of the chromosomal translocations t(14;18) and t(8;22) and in molecular genetic studies of transformed FL.     

Genomic alterations of the c-myc protooncogene in relation to the overexpression of c-erbB2 and Ki-67 in human breast and cervix carcinomas

Summary Genomic alterations of c-myc (amplification, rearrangements and hypomethylation) were investigated in 30 breast carcinomas and 20 cervix carcinomas. In breast carcinomas c-myc alterations were compared with overexpression of the c-erbB2 protooncogene and the proliferation marker Ki-67. Alterations of c-myc were found in 50% of the breast carcinomas and in 25% of the cervix carcinomas. In 23% of the breast carcinomas c-erbB2 overexpression was associated with c-myc alterations. In 17% of the cases there was overexpression of c-erbB2 without detectable alterations of c-myc. Hence, in 67% of breast cancers alterations of c-myc and/or c-erbB2 have been found, while in 81% of the samples Ki-67 expression was increased. The results suggest that the study of c-myc alterations provides an important complement to that of other prognostic indicators of breast cancer such as c-erbB2 expression.     

Reversal of multidrug resistance by novel cyclosporin A analogues and the cyclopeptolide SDZ 214-103 biosynthesized in vitro

It was shown that cyclopeptolide SDZ 214-103 (10 M) is more active in rhodamine-123 accumulation in actinomycin-d-resistant human lymphoma cells CCRF/ACTD400 than cyclosporin A (10 M), but equipotent in the doxorubicin-resistant Friend erythroleukemia cell line F4-6/ADR. In F4-6/ADR cells, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay showed comparable cytotoxic effects of doxorubicin at various concentrations in the presence of SDZ 214-103 and cyclosporin A. For the other novel cyclosporin A analogues minor multidrug-resistance-modulating potency was demonstrated. At equipotent modulating doses of verapamil (10 M) and cyclosporin A (10 M) in the MTT assay regarding doxorubicin cytotoxicity, cyclosporin A was efficient in the rhodamine-123-uptake assay while verapamil was not active when identical incubation times were used.Abbreviations MDR multidrug resistance - Pgp-170 P-glycoprotein with a molecular mass of 170 kDa - D-Hiv D-2-hydroxyisovaleric acid     

Nerve growth factor stimulates in vitro invasive capacity of DU145 human prostatic cancer cells

The prevalence of nerve growth factor (NGF) production in different human prostatic tumor cell lines (DU145, PC-3, LNCaP-FGC) was investigated using a specific enzyme-linked immunosorbent assay (ELISA) and compared to that of different human and rat prostatic tissue samples. In addition, the biological effects of NGF addition to the human prostatic cancer cell cultures were investigated. The ELISA technique showed the DU145 cell line to secrete measurable levels of NGF in the culture medium. When neurite-outgrowth determination in a pheochromocytoma cell line was used as a bioassay, the NGF synthesized by DU145 cells was confirmed to exhibit functional biological activity. No effect of exogenously added NGF could be established on tumor cell proliferation, on the basis of either colorimetric tetrazolium-based staining assay or bromodeoxyuridine incorporation. Also the expression of prostate specific acid phosphatase was not influenced by NGF addition. However, the in vitro invasive capacity (Matrigel) of DU145 cells was significantly increased by inclusion of 50 ng or 100 ng NGF/ml culture medium. In view of the clinically well-known perinuural invasion of prostate cancer cells, the possible involvement of NGF as a (paracrine) factor in prostatic cancer metastatic behavior should be investigated further.Abbreviations BSA bovine serum albumin - ELISA enzyme linked immunosorbent assay - NGF nerve growth factor - PBS phosphate-buffered salt solution - PSAP prostate-specific acid phosphatase     

Prognostic value of proliferating cell nuclear antigen and c-erbB-2 compared with conventional histopathological factors in breast cancer

Expression of proliferating cell nuclear antigen (PCNA) and c-erbB-2 oncoprotein has been assessed in 471 women with breast cancer to evaluate their prognostic value as compared to conventional histopathological factors. In univariate analysis, high PCNA expression (20%) predicted a significantly worse survival in lymph-node-negative tumors (univariateP=0.031). However, the effect disappeared in multivariate analysis and the histological grade remained the only independent factor for this group. Despite its close correlation to histological grade (P<0.001), PCNA expression discriminated subsets with different survival within the heterogeneous group of moderately differentiated tumors (univariateP=0.073, multivariateP=0.075). PCNA expression was not found to be a significant prognostic factor in lymph-node-positive tumors, thus it was of limited value for breast cancer patients as a whole. c-erbB-2 protein overexpression was associated with a worse survival (univariateP=0.019, multivariateP=0.057) for the entire group of patients. The effect was mainly attributed to the significance of c-erbB-2 as an independent factor in lymph-node-positive (up to three nodes, multivariateP=0.04; four or more nodes: multivariateP=0.017) and large tumors (>2 cm: multivariateP=0.002). c-erbB-2 was without significance in lymph-node-negative patients. Though both factors might amplify the prognostic information for distinct patient subsets they do not achieve the strong prognostic value of conventional histopathological features in breast cancer.     

Differentiation of Dunn osteosarcoma cells in response to dibutyryl cyclic 3′,5′-adenosine monophosphate

We investigated the effects of dibutyryl cyclic adenosine 3,5-monophosphate (Bt₂cAMP) on the differentiation of Dunn osteosarcoma cells. Flow-cytometric analysis and DNA synthesis assay showed that Bt₂cAMP decreased the cell population in the S phase in a time- and dose-dependent manner. Also, the cells showed distinct morphological and functional alterations: the cell morphology changed to a fibroblast-like appearance with long and thin protoplasmic processes, the knobs or blebs on both the cell membrane and nuclear membrane disappeared and the intracellular alkaline phosphatase activity increased. Moreover, Bt₂cAMP-treated cells secreted a large quantity of fibronectin, which was deposited on the extended cell surface in the culture medium. Thus, Dunn osteosarcoma cells are differentiated morphologically and functionally by Bt₂cAMP, and might be transformed to benign precursor cells.Abbreviation Bt₂cAMP dibutyryl cyclic 3,5-adenosine monophosphate This research was supported by a grant-in-aid for eucouragement of young scientists from the Japanese Ministry of Education, Science and Culture (02770900)     

Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification

Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (10⁵) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.Single-cell sequencing, characterization the genome of individual cells, is highly needed for studying scarce and/or precious cells, which are inaccessible for conventional bulk genome characterization, and for probing genomic variations of a heterogeneous population of cells (1–3). Recently single-cell genomics has unveiled unprecedented details of various biological processes, such as tumor evolution (4–6), embryonic development (7), and neural somatic mosaicism (8). Single-cell whole-genome amplification (WGA) is required to generate enough replicates of genomic DNAs for library preparation in conjunction with current sequencing protocols. Single-cell WGA has been increasingly used in cutting-edge clinical diagnostic applications such as molecular subtyping of single tumor cells (4, 9) and preimplantation genetic screening of in vitro fertilized embryos (10).An ideal single-cell WGA method should have high uniformity and accuracy across the whole genome. The WGA uniformity is critical for copy number variation (CNV) detection, whereas the WGA accuracy is essential for avoiding single-nucleotide variation (SNV) detection errors, either false positives or false negatives. The false positives arise from misincorporation of wrong bases in the first few cycles of WGA. In a diploid human cell, the false negatives primarily arise from the allelic dropout (ADO), i.e., heterozygous mutations are mistaken as homozygous ones because of the lack of amplification in one of the two alleles (11).Existing WGA chemistry includes degenerate oligonucleotide-primed PCR (DOP-PCR) (12), multiple displacement amplification (MDA) (13–17), and multiple annealing and looping-based amplification cycles (MALBAC) (4, 18, 19), which have successively achieved genome analysis at the single-cell level. DOP-PCR is based on PCR amplification of the fragments flanked by universal priming sites, and provides high accuracy for detecting CNVs in single cells but has low coverage and high false-positive and false-negative rates for calling SNVs (5). MDA has a much improved coverage but tends to have lower precision/sensitivity in CNV determination due to its variation of the amplification gain along the genome, not reproducible from cell to cell (20). By virtue of quasilinear amplification, MALBAC suppresses the random bias of amplification and exhibits reduced ADO rates, yielding low false negatives for SNV detection (2, 11, 18, 19). Notwithstanding its drawbacks, MDA still offers comparable or higher genome coverage than MALBAC, at least for single diploid cells, possibly taking advantage of the randomness (2). In fact, even higher coverage has been obtained for cells with aneuploidy, such as dividing cells (21), and cancer cells (22). MDA’s main advantage is its lower false-positive rate for SNV detection on account of the use of Phi-29, a highly processive polymerase with high fidelity.Microfluidic devices have been carried out for single-cell WGA (16, 20, 23, 24), allowing avoidance of contaminations and high-throughput analyses of multiple single cells in parallel. The small total reaction volumes (microliters to nanoliters- or picoliters) of the microfluidic devices not only facilitate the efficiency of reactions but also allow significant cost reduction for enzymes and regents used. It was reported that the nanoliter volume of a microfluidic device improved uniformity of the amplification compared with microliter devices in the WGA of single bacterial cells (20).Here, we report a method, emulsion whole-genome amplification (eWGA), to use the small volume of aqueous droplets in oil to better the WGA chemistry for uniform amplification of a single cell’s genome. By distributing single-cell genomic DNA fragments into a large number (10⁵) of picoliter droplets, a few DNA fragments in each droplet is allowed to reach saturation of DNA amplification. After merging the droplets by demulsification, the differences in amplification gain among the DNA fragments are significantly minimized.Although this approach can be used for any chemistry of WGA, we take MDA as an example to greatly reduce the random bias of amplification by separating the reactions into a large amount of emulsion droplets. We carried out detailed comparison with MDA, MALBAC, and DOP-PCR performed in tube using single cells from normal diploid human cells and a monoclonal human cancer cell line with inherited CNVs. Our results indicate that eWGA not only offers higher coverage but also enables simultaneous detection of SNVs and CNVs with higher accuracy and finer resolution, outperforming the prevailing single-cell amplification methods in many aspects.