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Biomedical subjects

Xuan Huang

Publications and source records attributed to Xuan Huang.

At least 19 recordsLinked to original sources
PubMed2026

Revealing potential biomarkers and metabolic mechanisms of ovarian aging in hens during late laying period based on machine learning and metabolomics.

Ovarian function decline during the late laying period represents a major bottleneck for the economic efficiency of the global poultry industry. However, the underlying metabolic mechanisms and reliable early-warning biomarkers for ovarian aging remain poorly understood. In this study, we performed the first untargeted LC-MS/MS metabolomics analysis of ovarian tissues from Taihe silky fowls at peak laying (30 weeks) and late laying (50 weeks) stages, and employed an ensemble machine learning strategy integrating LASSO, random forest, and support vector machine (SVM) algorithms to identify high-confidence core biomarkers of ovarian aging. Gene expression analysis was further conducted to validate the potential molecular mechanisms. Our results showed that the metabolic profiles of ovarian tissues differed significantly between the two groups. A total of 6 core biomarkers were identified, 4 of which were long-chain acylcarnitines. Mechanistic analysis revealed that downregulation of key genes in the carnitine shuttle system led to impaired mitochondrial fatty acid β-oxidation, which in turn triggered excessive oxidative stress and compromised ovarian endocrine function. In conclusion, this study identifies long-chain acylcarnitines as potential metabolic biomarkers for ovarian aging in Taihe silky fowls. These findings provide novel insights into the metabolic basis of poultry ovarian aging and lay a theoretical foundation for the precise regulation of reproductive performance in indigenous poultry breeds.

Animals↗
PubMed2023

Genome-wide identification of transcriptional enhancers during human placental development and association with function, differentiation, and disease†.

The placenta is a dynamic organ that must perform a remarkable variety of functions during its relatively short existence in order to support a developing fetus. These functions include nutrient delivery, gas exchange, waste removal, hormone production, and immune barrier protection. Proper placenta development and function are critical for healthy pregnancy outcomes, but the underlying genomic regulatory events that control this process remain largely unknown. We hypothesized that mapping sites of transcriptional enhancer activity and associated changes in gene expression across gestation in human placenta tissue would identify genomic loci and predicted transcription factor activity related to critical placenta functions. We used a suite of genomic assays [i.e., RNA-sequencing (RNA-seq), Precision run-on-sequencing (PRO-seq), and Chromatin immunoprecipitation-sequencing (ChIP-seq)] and computational pipelines to identify a set of >20 000 enhancers that are active at various time points in gestation. Changes in the activity of these enhancers correlate with changes in gene expression. In addition, some of these enhancers encode risk for adverse pregnancy outcomes. We further show that integrating enhancer activity, transcription factor motif analysis, and transcription factor expression can identify distinct sets of transcription factors predicted to be more active either in early pregnancy or at term. Knockdown of selected identified transcription factors in a trophoblast stem cell culture model altered the expression of key placental marker genes. These observations provide a framework for future mechanistic studies of individual enhancer-transcription factor-target gene interactions and have the potential to inform genetic risk prediction for adverse pregnancy outcomes.

Humans↗
PubMed2006

Constitutive histone H2AX phosphorylation and ATM activation, the reporters of DNA damage by endogenous oxidants.

DNA in live cells undergoes continuous oxidative damage caused by metabolically generated endogenous as well as external oxidants and oxidant-inducers. The cumulative oxidative DNA damage is considered the key factor in aging and senescence while the effectiveness of anti-aging agents is often assessed by their ability to reduce such damage. Oxidative DNA damage also preconditions cells to neoplastic transformation. Sensitive reporters of DNA damage, particularly the induction of DNA double-strand breaks (DSBs), are activation of ATM, through its phosphorylation on Ser 1981, and phosphorylation of histone H2AX on Ser 139; the phosphorylated form of H2AX has been named gammaH2AX. We review the observations that constitutive ATM activation (CAA) and H2AX phosphorylation (CHP) take place in normal cells as well in the cells of tumor lines untreated by exogenous genotoxic agents. We postulate that CAA and CHP, which have been measured by multiparameter cytometry in relation to the cell cycle phase, are triggered by oxidative DNA damage. This review also presents the findings on differences in CAA and CHP in various cell lines as well as on the effects of several agents and growth conditions that modulate the extent of these histone and ATM modifications. Specifically, described are effects of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), and the glutathione synthetase inhibitor buthionine sulfoximine (BSO) as well as suppression of cell metabolism by growth at higher cell density or in the presence of the glucose antimetabolite 2-deoxy-D-glucose. Collectively, the reviewed data indicate that multiparameter cytometric measurement of the level of CHP and/or CAA allows one to estimate the extent of ongoing oxidative DNA damage and to measure the DNA protective-effects of antioxidants or agents that reduce or amplify generation of endogenous ROS.

Acetylcysteine↗
PubMed2006

Activation of ATM and histone H2AX phosphorylation induced by mitoxantrone but not by topotecan is prevented by the antioxidant N-acetyl-L-cysteine.

The DNA topoisomerase II (topo2) inhibitor mitoxantrone (MXT) and topo1 inhibitor topotecan (TP) are antitumor drugs widely used to treat different types of cancer. Their mechanism of action is thought to stabilize otherwise transient ("cleavable") complexes between topo2 or topo1 and DNA; the collisions of the DNA replication fork during replication, or RNA polymerase during transcription, with these complexes convert them into double-strand DNA breaks (DSBs), potentially lethal lesions that may trigger apoptosis. In the present study we observed that treatment of human lung carcinoma A549 or promyelocytic leukemic HL-60 cells with MXT led to ATM activation and phosphorylation of histone H2AX on Ser-139, the reporters of induction of DSBs, in all phases of the cell-cycle. Only S-phase cells, however, underwent apoptosis after treatment with MXT, which implied that DSBs in the cells replicating DNA were more effective in triggering apoptosis than DSBs in G(1) or G(2)M phase cells. Unlike MXT, the treatment with TP induced ATM activation and H2AX phosphorylation almost exclusively in S-phase cells and only S phase cells underwent apoptosis. The induction of both ATM activation and H2AX phosphorylation by MXT was prevented to a large extent by N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS). The protective effect of NAC was observed for cells in all phases of the cell cycle. NAC offered no protection at all against TP. The induction of DSBs by MXT, thus, appears to be predominantly mediated through ROS, while DSBs generated during treatment with TP most likely are a consequence of collisions of replication forks with the "cleavable" complexes.

Acetylcysteine↗
PubMed2006

Nitrogen oxide-releasing aspirin induces histone H2AX phosphorylation, ATM activation and apoptosis preferentially in S-phase cells: involvement of reactive oxygen species.

Nitric oxide-releasing acetylsalicylic acid (NO-ASA; NO-aspirin) developed as an anti-inflammatory agent that was expected to avoid some of the adverse effects of aspirin (ASA), was recently shown to be cytotoxic to cells of different tumor lines. The cytotoxic properties and potency of NO-ASA are different than those of ASA which implies that the intracellular targets for NO-ASA and ASA, and their mechanism of action, are different. The aim of the present study was to reveal whether the cytotoxicity induced by NO-ASA is mediated by damage to DNA. We observed that even brief (1 h) treatment of human B-lymphoblastoid TK6 cells with >or=5 microM NO-ASA led to DNA damage revealed by the alkaline and neutral comet assays, histone H2AX phosphorylation on Ser 139, and ATM phosphorylation on Ser 1981, a marker of activation of this kinase. The induction of H2AX phosphorylation was preferential to S-phase cells. Exposure to >or=5 microM NO-ASA for over 3 h led to apoptosis, also preferentially of S-phase cells. Apoptosis was atypical; while chromatin was highly condensed there was no evidence of nuclear fragmentation nor were the cells positive in the TUNEL assay though they did express activated caspase-3. The induction of phosphorylation of H2AX on Ser 139 by NO-ASA was markedly attenuated in the presence of N-acetyl-L-cysteine, a scavenger of reactive oxygen species (ROS). The data imply that the NO-ASA induces DNA damage through oxidative stress; the oxidation-generated lesions provide a signal for induction of H2AX phosphorylation during DNA replication, perhaps when the progressing replication forks collide with the primary lesions converting them to DNA double-strand breaks. Because neither induction of H2AX phosphorylation nor apoptosis were observed at equimolar concentrations of ASA, the NO moiety attached to ASA appeared to mediate these effects.

Apoptosis↗
PubMed2006

Significance of Survivin and PTEN expression in full lymph node-examined gastric cancer.

AIM: To study the relationship between Survivin and PTEN expression and lymph node metastasis, depth of invasion and prognosis of gastric cancer patients in China. METHODS: Specimens of gastric cancer tissue were collected from the Affiliated Hospital of Jianghan University. All the 140 patients had complete examination data. All lymph nodes were found by the fat-clearing method. The interrupted serial 4-micron sections, routine hematoxylin and eosin staining and immunohistochemical methods were used to detect the lymph node metastases. Gastric cancer tissue microarray was performed to test the expression of Survivin and PTEN (17A) in gastric cancer by immunohistochemical method. All data were processed using chi2 test, Fisher's exact test, Kaplan-Meyer Log-rank method and Cox multivariate analysis (SPSS 12.0 software). RESULTS: One hundred and eighteen specimens were used in our tissue microarray (utilization rate was 82.4%). A total of 7580 lymph nodes were found. Metastases were found in 90 specimens and 1618 lymph nodes were detected. The positive rate of Survivin and PTEN expression was 52.5% (62/118) and 76.2% (90/118), respectively. A highly positive correlation was found between Survivin and PTEN expression (chi2 = 4.17, P = 0.04). Survivin expression was positively correlated with UICC N stage (chi 2 = 8.69, P = 0.03) and histological classification (chi2 = 4.41, P = 0.04) by chi2 tests. PTEN expression was positively correlated with depth of invasion (P = 0.02) and histological classification (chi2 = 5.47, P = 0.02). But Survivin and PTEN expressions were not related with prognosis of gastric cancer patients. A significant correlation between lymph node metastasis and prognosis was demonstrated by Cox multivariate analysis (chi2 = 4.85, P = 0.028). CONCLUSION: Survivin is positively correlated with PTEN expression in gastric cancer and is a molecular marker of lymph node metastasis while PTEN expression is a molecular marker of advanced gastric cancer. UICC N stage is the most important prognostic factor of gastric cancer in China.

Adult↗
PubMed2006

Parental weight status as a moderator of the relationship between television viewing and childhood overweight.

OBJECTIVE: To examine whether and to what extent the relationship between television viewing and children's weight status differs according to parental weight status. DESIGN: Population-level survey including in-home and telephone interview components. SETTING: United States. PARTICIPANTS: Representative sample of children aged 6 to 19 years in 2002 (n = 1483). MAIN EXPOSURE: Hours of television viewing. MAIN OUTCOME MEASURES: Child weight status (normal weight, at risk for overweight, overweight) as defined by current Centers for Disease Control and Prevention standards. RESULTS: Parental obesity increased the risk of child overweight for all of the children except boys aged 6 to 9 years. There were significant interactions between television viewing hours and parental obesity among boys aged 14 to 19 years and girls aged 10 to 13 years. For these 2 groups, the odds of overweight status increased with viewing hours for children with at least 1 obese parent but not at all for children of normal-weight parents. CONCLUSIONS: Results indicate that when parental obesity is taken into account, television viewing hours do not significantly relate to increased odds of childhood overweight, and parental body mass index may serve to moderate the relationship between television viewing and child weight status among adolescents (but not among younger children). Further examination of the moderating effect of parental body mass index on the relationship between television viewing and child weight status is warranted.

Adolescent↗
PubMed2006

Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis.

BACKGROUND: DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of alpha-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM. MATERIALS AND METHODS: HL-60 cells were treated in cultures with 0.1-5.0 mM HU or 1-4 muM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content. RESULTS: While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1-1.4) were more affected than late-S phase (DI = 1.6-1.9) cells, ATM was not activated by HU. In fact, the level of constitutive ("programmed") ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1-5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation. CONCLUSIONS: The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive ("programmed") level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation.

Aphidicolin↗
PubMed2006

Sequential phosphorylation of Ser-10 on histone H3 and ser-139 on histone H2AX and ATM activation during premature chromosome condensation: relationship to cell-cycle phase and apoptosis.

BACKGROUND: Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to cell-cycle phase and to initiation of apoptosis. MATERIALS AND METHODS: To induce PCC, A549 and HL-60 cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of cellular fluorescence was measured by flow- or laser scanning cytometry. RESULTS: Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 cells. All these events occurred sequentially, prior to caspase-3 activation, and affected cells in all phases of the cell cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 cells. CONCLUSIONS: Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some cell types may proceed without ATM activation and H2AX phosphorylation.

Apoptosis↗
PubMed2006

Cytometric assessment of histone H2AX phosphorylation: a reporter of DNA damage.

DNA damage that leads to formation of DNA double-strand breaks (DSBs) induces phosphorylation of histone H2AX on Ser-139 at sites flanking the breakage. Immunocytochemical detection of phosphorylated H2AX (denoted as gammaH2AX) thus provides a marker of DSBs. The method presented in this chapter describes the detection of gammaH2AX for revealing the presence of DSBs, combined with differential staining of cellular DNA for revealing the cell cycle phase. The detection of gammaH2AX is based on indirect immunofluorescence using secondary antibody tagged with fluorescein isothiocyanate (FITC) while DNA is counterstained with propidium iodide (PI). Intensity of cellular green (FITC) and red (PI) fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of DSBs with the cell cycle phase.

Animals↗
PubMed2006

Detection of DNA strand breaks by flow and laser scanning cytometry in studies of apoptosis and cell proliferation (DNA replication).

Extensive fragmentation of nuclear DNA occurs during apoptosis, and the presence of DNA strand breaks is considered to be a marker of the apoptotic mode of cell death. This chapter describes methods to label in situ DNA strand breaks with fluorochromes for detection by flow or laser scanning cytometry. By staining DNA with a fluorochrome of another color, cellular DNA content is measured concurrently and the bivariate analysis of such a data reveals DNA ploidy and cell-cycle phase position of apoptotic cells. The DNA strand break-labeling methodology is also used for detecting the incorporation of halogenated DNA precursors in studies of the cell cycle, proliferation, and DNA replication. In this application, termed "strand breaks induced by photolysis" (SBIP), the cells are incubated with 5-bromo-2'-deoxyuridine (BrdU) to incorporate it into DNA and sensitize the DNA to ultraviolet (UV) light. DNA strand breaks are then photolytically generated by exposing the cells to UV light. The DNA strand breaks resulting from UV-photolysis are subsequently fluorochrome-labeled as for labeling apoptotic-DNA breaks. Because SBIP, unlike the alternative method of detection of BrdU incorporation, does not require subjecting cells to harsh conditions (strong acid or heat) of DNA denaturation, it is compatible with concurrent detection of intracellular or cell surface antigens by immunocytochemical means.

Apoptosis↗
PubMed2006

Induction of DNA double-strand breaks in A549 and normal human pulmonary epithelial cells by cigarette smoke is mediated by free radicals.

DNA double-strand breaks (DSBs) are potentially mutagenic/carcinogenic lesions. Induction of DSBs triggers phosphorylation of histone H2AX on Ser-139. Phosphorylated H2AX (gammaH2AX) can be detected immunocytochemically, and the intensity of gammaH2AX immunofluorescence (IF), reflecting the number of gammaH2AX-IF foci per nucleus, reveals the frequency of DSBs. Using multiparameter cytometric analysis of gammaH2AX-IF, we previously observed that DSBs are induced in normal human bronchial epithelial (NHBE) and A549 pulmonary adenocarcinoma cells following exposure to cigarette smoke (CS) or smoke condensate. In the present study, we show that N-acetyl L-cysteine (NAC) and glutathione, both effective scavengers of free radicals, prevented induction of DSBs by CS in these cells. In contrast, the glutathione synthesis inhibitor, DL-Buthionine-[S,R]-sulfoximine (BSO), enhanced the induction of DSBs by CS. The observed reduction of DSBs by NAC correlated with protection of the reproductive capability (clonogenicity) of A549 cells treated with CS. The data implicate formation of free radicals by CS as factors generating DSBs and affecting cell survival. Interestingly, at the conditions of exposure to CS when clonogenicity was only moderately affected, S-phase cells showed significantly higher sensitivity in terms of induction of DSBs compared with G1 or G2M cells. In light of the evidence that CS increases oxidative stress and induces cell proliferation in the lungs of smokers, the high propensity of S-phase cells to develop DSBs upon exposure to CS has to be considered as a potentially pathogenic event in smoke-induced tumor development. This is the first report to reveal cell cycle-phase specificity in both the induction of DSBs by CS and their prevention by free radical scavengers. The detection of gammaH2AX to assess the induction of CS-induced DSBs and their relationship to cell cycle phase provides a convenient tool to explore approaches to protect cells from this type of genotoxic damage.

Cell Line, Tumor↗
PubMed2006

Constitutive histone H2AX phosphorylation on Ser-139 in cells untreated by genotoxic agents is cell-cycle phase specific and attenuated by scavenging reactive oxygen species.

DNA damage, particularly when it involves formation of double-strand breaks (DSBs), triggers phosphorylation of histone H2AX on Ser-139. Phosphorylated H2AX has been named gammaH2AX, and induction of gammaH2AX in cells exposed to genotoxic agents is considered a sensitive and specific reporter of DNA damage. However, in untreated normal cells as well in the cells of various tumor lines cells, a fraction of histone H2AX molecules remain phosphorylated. In the present study, we observed that the extent of this constitutive H2AX phosphorylation varies depending on the cell type (line) and on cell cycle phase and, in most cell types, S and G(2)/M phase cells exhibit greater levels of H2AX phosphorylation than do cells in the G(1) phase. Furthermore, constitutive H2AX phosphorylation in human pulmonary carcinoma A549, lymphoblastoid TK6, and in normal bronchial epithelial cells was reduced following cell exposure to N-acetyl-L-cysteine, a scavenger of reactive oxygen intermediates; the reduction was most pronounced for G(2)M cells. Growth of A549 cells in the presence of buthionine sulfoximine, an inhibitor of glutathione synthetase, amplified the level of constitutive H2AX phosphorylation in A549 cells. The observed constitutive H2AX phosphorylation may be a reflection of the ongoing DNA damage mediated by reactive oxygen species (ROS) generated by metabolic activity during progression through the cell cycle, leading to formation of DSBs during the S phase. Because cumulative DNA damage in proliferating cells mediated by ROS is considered the key mechanism for cell ageing, the present approach to estimate the degree of attenuation of constitutive H2AX phosphorylation by antioxidants may provide a convenient tool to assess the DNA-protective and possible anti-ageing properties of other agents.

Antineoplastic Agents↗
PubMed2006

[Cloning and identification of the resistance gene analogs of wheat].

Specific primer sequences were designed on the basis of converse motifs of cloned resistance genes and subsequently used as PCR primers to amplify the resistance gene analog (RGA) in wheat (Triticum aestivum L.) Xinong 88 which possesses rust disease resistance. The amplified products of WRGA1, WRGA2 and WRGA14 were cloned and sequenced. The wheat RGAs contain some conserved motifs such as Kinase-2a, Kinase-3a and HD presenting in known NBS-LRR resistance genes from other plants and share between 46.0%-9.9% amino acid sequence identity with them. The amino acid sequence identity among three RGAs is 80.7%-56.8%. The WRGA1 expression was induced and detected by Northern blotting, which is positively regulated by salicylic acid.

Amino Acid Sequence↗
PubMed2005

Contribution of p16INK4a and p21CIP1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53.

We have previously found that nonenzymatically glycated collagen I (GC), mimicking diabetic microenvironment, can induce senescent phenotype in early passage human umbilical vein endothelial cells (HUVECs). In the present study, we explored the functional involvement of cell cycle checkpoint pathways in initiating GC-induced premature endothelial cell senescence. When compared with native collagen, early passage HUVECs showed increased p53, p21(CIP1) (p21), and p16(INK4a) (p16) mRNA expression after exposure to GC. Twenty-four hours after transfection of p16, p21, and p53-enhanced green fluorescent protein (EGFP) recombinant plasmids, HUVECs entered G(1)-phase cell cycle arrest. By days 3 and 5, HUVECs transfected with p16-EGFP showed an increased proportion of senescent cells, and this increase was more prominent in the GFP-positive cell population, which exhibited 68% of senescent cells. Transfection of p21 also induced senescence but only by day 5. Cotransfection of p16 and p21 showed no additive effect. Transfection of p21 or p53 induced apoptosis in HUVECs. Next, we suppressed endogenous p53, p21, p16, or retinoblastoma (Rb) gene expression through small interference RNA strategy and investigated their influence in p16- and p21-initiated endothelial cell senescence. Analysis indicated that suppression of p53 expression can abolish senescence induced by p16 overexpression. Paradoxically, this effect was not observed when p21 was suppressed. On the other hand, suppression of Rb eliminated senescence initiated by either p16 or p21 overexpression. In summary, the p53/p21 pathway is mainly responsible for GC-induced apoptosis, but the coordinated activation of the p53/p21 and p16 pathway is responsible for GC-induced endothelial cell senescence through a Rb-dependent mechanism.

Animals↗
PubMed2005

Enforced adhesion of hematopoietic cells to culture dish induces endomitosis and polyploidy.

Cells of epithelial or endothelial lineage when forced to grow in suspension undergo the detachment-induced death termed "anoikis". In the present study we explored the reverse situation namely the effect of enforcement of hematopoietic lineage cells that are normally maintained in suspension to grow attached. Towards this end murine L1210 or human HL-60 and Jurkat leukemia cells were cultured in slide chambers coated with poly-L- or poly-D- lysine, or with compound 48/80, the polycations attracting them electrostatically. Within minutes after the transfer L1210 cells strongly adhered to bottom surface of the dish and shortly thereafter binuclear-, and later on, polynuclear-cells become apparent. The frequency of nuclei per cell was increasing with time and polykaryons with up to 16 nuclei and high DNA ploidy (DI = 16.0) were apparent after 48 h. After 4 days the size (volume) of some polykaryons exceeded by over 340-fold the volume of mononuclear cells. The presence of mitotic figures and abnormal mitotic spindles in adhering polykaryons provided evidence of the impeded cytokinesis that led to endomitosis. Most polykaryons excluded trypan blue, had balanced growth (unchanged protein/DNA ratio compared to monokaryons), and showed no evidence of apoptosis. Individual nuclei within each polykaryon replicated DNA in synchrony. The strong cell attachment and aborted cytokinesis were cell line specific since no significant endomitosis was observed in Jurkat- or HL-60- cells which did not strongly attach to polycation-coated surfaces. Defective cytokinesis and induction of polyploidy by this mechanism, if occurs in vivo (e.g., mediated by integrins), may lead to aneuploidy and therefore have tumorigenic consequences. The phenomenon offers novel experimental model for induction of polyploidy and provides alternative to cytocholasin B to prevent cytokinesis in the mutagenicity cytokinesis-blocked micronucleus (CBMN) assay.

Animals↗