Molecular biology of astroviruses: selected highlights.

Human astrovirus, the prototype of the Astroviridae family, is a non-enveloped positive-strand RNA virus with distinctive morphology. Initially named for a characteristic 5-6 point star evident on the surface of faecally shed viral particles by direct electron microscopy, a recent study using cryoelectron microscopy and image reconstruction indicates that viral particles consist of a smoothly rippled, solid capsid decorated with short spikes. Mechanisms underlying the assembly of these viral particles have not been fully elucidated. However, studies of two full-length cDNA clones of human astrovirus serotype 1 suggest that capsid residue Thr227 plays a critical role in the assembly of infectious viral progeny. The development of a full-length clone (pAVIC) from which infectious RNA can be transcribed has also facilitated studies of the viral 3C-like serine protease, encoded in ORF1a. These studies demonstrate that the full-length ORF1a product (101 kDa) is processed in vitro to an N-terminal 64 kDa fragment and a C-terminal 38 kDa fragment. Mutation of the predicted catalytic triad inhibits proteolysis. In other studies based on modifications of pAVIC, preliminary evidence supports the feasibility of developing a reporter cell line to facilitate astrovirus detection.

Involving AP-2 transcription factor in connexin 26 up-regulation during pregnancy and lactation.

Gap junction connexin 26 (Cx26) is up-regulated in mammary epithelial cells during pregnancy and lactation. To understand the transcriptional regulation of Cx26, we identified a protected DNase I footprint region (-140 to -113) in the rat Cx26 promoter. This rCx26 Promoter Footprinting Region, or CPFR, contains an Sp binding site (CCGCCC) overlapping with an AP-2 binding site (GCCCGCGGC), and is evolutionarily conserved. Nuclear extracts from rat mammary glands and human MCF-10 mammary epithelial cells formed protein-DNA complexes with the labeled CPFR probe in the electrophoretic mobility shift assay (EMSA), and these complexes were markedly enhanced during pregnancy and lactation. Antibody supershift analysis further identified the presence of Sp1, Sp3, and AP-2 in these binding complexes. Human mammary epithelial MCF-10A and MCF-12A cells were transiently transfected with chimeric mutant rCx26 promoter/luciferase reporter constructs, and luciferase activities measured. Mutations along the CPFR fragment drastically reduced the promoter activity, specially at the Sp/AP-2 overlapping site. Cotransfection of AP-2 with rCx26 promoter/reporter constructs into MCF-10 cells markedly induced the reporter activity. These data infer that AP-2, along with previously reported Sp transcription factors, is involved in the up-regulation of Cx26 gene during pregnancy and lactation.

Detection rate of Actinobacillus actinomycetemcomitans on the permanent 1st molars of primary school children in Taiwan by polymerase chain reaction.

BACKGROUND, AIMS: Actinobacillus actinomycetemcomitans (Aa) has been implicated as the putative micro-organism for localized juvenile periodontitis (LJP). The most distinct clinical features of LJP include severe angular bony defects of the mesial sides of permanent first molars and the onset of disease during puberty. Currently, no large-scale studies have been performed which address the change in detection rates of Aa on the mesial sides of permanent 1st molars following eruption and up to puberty. METHOD: In this study, subgingival plaque samples were taken from the mesial pockets of 2 randomly selected permanent 1st molars from 328 primary school children and 50 adult staff, and analyzed by polymerase chain reaction (PCR) to detect Aa. RESULTS: The results showed a 5.5% prevalence rate of Aa which increased after the eruption of 1st molars and peaked near puberty. There were no significant differences in the detection rates of Aa among different groups in terms of gender, plaque index (PII), and gingival index (GI); however, the higher detection rates of Aa were significantly associated with increased probing depths at p<0.05. CONCLUSION: PCR analysis of the subgingival plaques demonstrated a prevalence of Aa which peaked near puberty, suggesting that Aa may be important for LJP in Taiwan.

An outbreak of enterovirus 71 infection in Taiwan, 1998. II. Laboratory diagnosis and genetic analysis.

BACKGROUND: An epidemic of enterovirus 71 (EV71) occurred in Taiwan from April to December of 1998, with two peaks, one in June and the other in October. Many enteroviruses were isolated in our laboratory from 258 cases during this outbreak. Approximately half of the enteroviruses isolated were EV71 and one fifth were coxsackievirus A16. OBJECTIVES: To analyze laboratory findings in the EV71 epidemic of 1998 in Taiwan, various EV71 specimens in different cell lines were examined. In addition, genetic analysis of 5' non-coding region (NCR) was performed to analyze the strain variation in this outbreak. RESULTS: The cytopathic effect induced by EV71 was observed 2-13 (mean of 4.5) days post-inoculation in Vero cells and 4-15 (mean of 6.6) days in green monkey kidney (GMK) cells inoculated with throat swabs. Of the total positive EV71 cases, virus was most frequently obtained from throat swabs (91.7%), less from stools (64.8%), and none from cerebral spinal fluid (CSF). Molecular analyses of EV71 by sequencing the 5' NCR of 34 strains obtained from different clinical categories and various geographic areas showed that their sequences differed (0-13 bp in 681 bp sequenced) by approximately 0-2%. The sequences of these isolates differed from EV71 prototype BrCr or MS strain by 17.5-19%, with the exception of two samples which exhibited nucleotide variation by only 8.9 and 8.2%, when compared to the MS strain. CONCLUSION: EV71 was most frequently isolated from throat swab specimens in Vero cells. The molecular analyses of the 5' NCR of EV71 revealed that most isolates from this epidemic belonged to a group of closely related clones and only two were in a different group which was clustered with the EV71 MS strain.

Modulation of the connexin26 tumor suppressor gene expression through methylation in human mammary epithelial cell lines.

BACKGROUND: Normal mammary epithelial cells express mainly gap junction connexin 26 (Cx26) that is either reduced or absent in breast cancers. Since connexin gene mutations are rare we examined if Cx26 gene repression is related to hypermethylation. MATERIALS AND METHODS: Five breast epithelial cell lines were examined for Cx26 mRNA expression and hypermethylation. Treatment with a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (5-Aza-CdR), was carried out to determine if Cx26 gene expression could be upregulated. RESULTS: Cx26 expression was easily detectable in an immortalized human mammary epithelial cell line (MCF-10) and markedly diminished (MDA-MB231) or undetectable in (MCF-7, BT-20, T47-D) breast cancer cell lines. Hypermethylation of the Cx26 5' region was observed in MCF-10 and MCF-7 cells. Treatment with 5-Aza-CdR resulted in slight or no induction in Cx26 expression in breast cancer cell lines. CONCLUSIONS: Hypermethylation is unlikely to be a major mechanism for Cx26 gene repression in human mammary cancer cell lines.

Differential up-regulation of gap junction connexin 26 gene in mammary and uterine tissues: the role of Sp transcription factors.

The mRNA and protein expressions of connexin 26 (Cx26) in rat mammary gland and uterus can be up-regulated during pregnancy as well as by the administration of human CG (hCG). In the present study, we found that the time course and magnitude of Cx26 induction by hCG was different in these two tissues. The molecular mechanism underscoring this difference was therefore investigated. We had previously demonstrated that both Sp1 and Sp3 transcription factors play a functional role in Cx26 expression. By the electrophoretic mobility shift assay, nuclear extracts from both virgin mammary gland and uterus were capable of binding to a labeled oligonucleotide probe that contained the proximal GC box and formed three protein-DNA complexes (C1, C2, and C3). In the mammary gland, pregnancy enhanced the intensity of all three complexes, whereas in the uterine tissue there was a decrease in the C2 and C3 complexes and an emergence of a new major component, C4 complex. In the supershift study, the C1 complex could be supershifted only by an antibody against Sp1, whereas C2, C3, and C4 could all be supershifted by an antibody against Sp3, suggesting a potential presence of Sp3 isoforms of various sizes. We therefore conclude that the basal Sp profiles in virgin mammary gland and uterine tissue are similar. However, in response to pregnancy, the changes in Sp profile are tissue specific and may account for the temporal and quantitative differences between these two tissues in Cx26 induction.

Mapping and characterization of the basal promoter of the human connexin26 gene.

Connexin26 (Cx26) is a major gap junction protein expressed in mammary and endometrial epithelial cells. Previously, we have cloned the genomic upstream sequence of the human connexin26 gene. In this paper, we studied the structure and function of its basal promoter. Various 5'-flanking regions of the human Cx26 gene were inserted upstream of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and transfected into human immortalized mammary MCF-10A and MCF-12A cell lines and endometrial RL95-2 cancer cell line. Through CAT reporter gene analysis, we identified the basal promoter of human Cx26 gene in the proximal 5'-flanking region from -128 to +2 (relative to the transcription initiation site). Further deletion analyses suggested that the critical regulatory area was located within a 29 bp region (from -97 to -69), where two GC consensus boxes (CCGCCC) resided, one at -93 and the other at -81. Labeled oligonucleotides encompassing these two GC box DNA sequences could bind the nuclear extracts from MCF-12A and RL95-2 cells in the electrophoretic mobility shift assay. These binding complexes could be competitively reduced by non-labeled self or Sp1 consensus oligonucleotide, and supershifted by antibodies against either Sp1 or Sp3. Mutations in the core sequence of these two GC boxes from CCGCCC to CCGAAC caused a loss of competitive ability and also produced a drastic reduction of basal promoter activity when integrated into promoter/reporter constructs. Furthermore, co-transfection of Sp1 and/or Sp3 expressing plasmids could trans-activate the expression of human Cx26 promoter/reporter constructs in Drosophila Schneider line 2 (SL2) cells. Taken together, these data indicated that the two GC boxes in the proximal promoter region play an important role in the control of human Cx26 gene expression.

Gap junction Cx26 gene modulation by phorbol esters in benign and malignant human mammary cells.

Connexin (Cx) 26, a major gap junction protein expressed in mammary epithelial cells, has been considered to be a tumor suppressor gene candidate. This study investigated the molecular mechanism of transcriptional up-regulation of Cx26 by phorbol ester (TPA) in human immortalized MCF-10 mammary epithelial cells and MDA-MB-231 mammary cancer cells. Such up-regulation was mediated through the protein kinase C pathway and could be blocked by the PKC inhibitor, calphostin C. Based on the results of the nuclear run-on assay, there was a TPA-induced increase in the rate of transcriptional initiation. We identified a TPA-induced DNase I hypersensitivity (DH) region approximately 1 kb 5' upstream of the ATG translation starting site. Sequence analysis revealed that this DH region was located in intron 1 and contained two TRE-like TGAT/ATCA elements, two 5'TTCA3' motifs and a 5'AGGAAG3' PEA3 motif. Both TRE-like elements were capable of binding AP1. TPA inducibility of this DH region was seen by the CAT reporter assay and appeared to be direction-dependent suggesting a functional cooperation between PEA3/TTCA and TRE.

Direct modulation of tumor suppressor connexin 26 gene by human chorionic gonadotropin in rat mammary glands.

Human chorionic gonadotropin (hCG) has been shown to reduce the incidence of carcinogen-induced rat mammary tumors. Because connexin 26 (Cx26), a tumor suppressor gene candidate, can be up-regulated in mammary epithelial cells during lactation, we examined the in vivo and ex vivo effects of hCG on Cx26 expression in rat mammary tissues and used its effect on the expressions of beta-casein and Cx43 as controls. The Cx26 mRNA and protein expressions were up-regulated by daily administrations of 100 units of hCG, starting on day 5 and reaching a 14-fold maximum increment on days 16 through 21. It remained elevated above the basal level even 20 days after hCG withdrawal. The changes in beta-casein expression ran parallel to that of Cx26, whereas the expression of Cx43 was down-regulated. There was no correlation between steroidal hormone levels and Cx26 expression, except for the first 5 days of hCG treatment. In the ex vivo organ culture system, exposure of mammary glands to 10 units/ml hCG for 5 days up-regulated Cx26 but had no effect on beta-casein expression. These results imply a direct induction of the tumor suppressor Cx26 gene by hCG in mammary epithelial cells, a mechanism unrelated to lactation.

Upstream genomic sequence of the human connexin26 gene.

Human connexin 26 (Cx26) has been considered to be a candidate suppressor gene in mammary epithelial cells. To gain insight into the transcriptional regulation of this gene, we have cloned and sequenced the 5' portion of the gene, which extends 4.8 kb upstream from the ATG translation start site. The 3' end of the non-coding exon 1 (160 bp) is located at 3149 bp upstream from the 5' end of exon 2. Comparison between the human Cx26 gene and the mouse gene reveals a highly conserved promoter region with 81% homology. In addition to six GC boxes and two GT boxes, a TTAAAA box is located at -24 to -19 bp upstream of the transcription start point. Analogous to the mouse beta-casein gene, the promoter region of the human Cx26 gene also contains a YY1-like binding site and a consensus mammary gland factor binding site.

Use of Truquant BR radioimmunoassay for early detection of breast cancer recurrence in patients with stage II and stage III disease.

PURPOSE: The Truquant BR radioimmunoassay (RIA) (Biomira Diagnostics Inc, Rexdale, Canada) uses the monoclonal antibody B27.29 to quantitate the MUC-1 gene product (CA 27.29 antigen) in serum. We evaluated CA 27.29 antigen in a controlled, prospective clinical trial for its ability to predict relapse in stage II and stage III breast cancer patients. PATIENTS AND METHODS: Over a 2-year period, 166 patients who had completed therapy for stage II (80.1%) or III (19.9%) breast cancer and were clinically free of disease were serially tested for CA 27.29 antigen levels. The study was double-masked and cancer recurrence was documented based on clinical findings. Patients with two consecutive CA 27.29 antigen test results above the upper limit of normal were considered positive. RESULTS: The Truquant BR RIA had a sensitivity of 57.7%, specificity of 97.9%, positive predictive value of 83.3%, and negative predictive value of 92.6%. The recurrence rate was 15.7%. A Cox regression analysis showed that the only variable to correlate with recurrent disease was the CA 27.29 antigen test result. Patients with a positive test result had increased odds of having a recurrence (odds ratio, 6.8; P < .00001). The test was effective in predicting recurrence in patients with both distant and locoregional disease. In a subgroup of patients with bone pain, CA 27.29 antigen level was found to identify reliably patients who would subsequently develop recurrent disease. CONCLUSION: These data demonstrate that the Truquant BR RIA can be used as an aid to predict recurrent breast cancer in patients with stage II and III disease.

Psychological symptoms and disease-free and overall survival in women with stage II breast cancer. Cancer and Leukemia Group B.

BACKGROUND: The possible link between psychological factors and length of cancer survival has generated a literature of contradictory findings. Associations usually have not been found when general psychological symptoms are assessed. Associations usually have been found for predictors related to expressive versus repressive emotional coping (e.g., depression, "fighting spirit," hostility, and type C personality); however, even these associations have been relatively small, when compared with those for medical factors. Yet few studies have adequately controlled for medical and treatment-related factors. PURPOSE: Within a Cancer and Leukemia Group B (CALGB) national clinical trial of four adjuvant therapy regimens for stage II breast cancer (CALGB 8082), this study prospectively examined the contribution of potential psychological predictors to length of disease-free and overall survival over a 15-year period. METHODS: Subjects were 280 women with stage II breast cancer, out of a total of 899, who were randomly assigned to receive CMFVP (cyclophosphamide-methotrexate-fluorouracil-vincristine-prednisone) for two 6-week cycles or six 4-week cycles, then subsequently randomly assigned to receive or not to receive VATH (vinblastine-doxorubicin-thiotepa-fluoxymesterone). Subjects were recruited during the period between October 1980 and August 1984, inclusive, and followed until January 1996. Prior to chemotherapy, psychological symptoms were assessed using the Symptom Check List-90-Revised (SCL-90-R). SCL-90-R scores were trichotomized into categories representing high, medium, and low distress. Basic base-line sociodemographic data (including age, ethnicity, education, and marital status) and medical data (including lymph node status, estrogen receptor status, menopausal status, and performance status) were collected. Subjects with psychosocial data differed from those without psychosocial data solely in their higher percentage of classification in the mild limitation category of the Zubrod (Eastern Cooperative Oncology Group) performance status rating (subjects with psychosocial data: 14%; subjects without psychosocial data: 8%). RESULTS: In stepwise Cox regression analyses that controlled for sociodemographic and medical variables, there was no significant predictive effect of the level of distress (as measured by the SCL-90-R trichotomized scores) on length of disease-free and overall survival of the study subjects. Risk ratios for low versus high distress were 1.01 (95% confidence interval [CI] = 0.62-1.66) for disease-free survival and 1.03 (95% CI = 0.58-1.82) for overall survival. CONCLUSIONS: This study failed to provide evidence that psychological factors contributed to length of disease-free or overall survival of women who received adjuvant chemotherapy (either CMFVP alone or CMFVP followed by VATH) for treatment of stage II breast cancer. IMPLICATIONS: In the context of far more potent medical factors, the contribution of psychological factors to disease-free and overall survival is likely to be relatively small. Future research should focus on specific theory-driven predictors rather than on general psychological symptoms. Moreover, it should be based on clinical studies using a controlled, prospective design, in which the effects of medical factors may be distinguished and psychological predictors are clear antecedents of survival outcomes.

Cloning the gene encoding Schistosoma mansoni p50, an immunophilin.

A 2.2-kb fragment of genomic DNA encoding Schistosoma mansoni immunophilin p50 (Smp50) was identified on a 14-kb genomic clone. The sequence of Smp50 reveals seven exons interrupted by six small introns ranging from 28-35 bp in size. The transcription start point, defined by primer extension analysis of schistosome RNA, begins at 30 bp upstream from the start AUG codon. Smp50 lacks a TATA box and appears to be a single-copy gene.

Identification and characterization of Schistosoma mansoni p17.7, a cyclophilin.

Antibodies affinity purified against tegumental components of schistosomula were used to screen a Schistosoma mansoni lambda gt11 adult worm cDNA expression library. One of the reactive clones was determined by sequence analysis to encode a protein homologous to cyclophilins of other species, in particular cyclophilin A. The 0.8-kb cDNA clone contained an open reading frame of 483 nucleotides which corresponds to a translation product of 161 amino acids with a deduced molecular size of 17.7 kDa. We have chosen to designate this clone as S. mansoni p17.7 (Smp17.7). The overexpressed and purified recombinant Smp17.7 (rSmp17.7) was demonstrated to possess peptidylprolyl cis-trans isomerase (PPIase) or rotamase activity typical of cyclophilins. Western blot analysis of Nonidet P-40 and a total soluble extract of adult schistosomes probed with affinity-purified antisera to rSmp17.7, demonstrated the presence of this protein in the parasite. Immunofluorescence studies using the purified antisera indicates a localization in various tissues including the tegument and the gut. As cyclophilin is able to interact with cyclosporin A (CsA), which has been shown to be antischistosomal in mice infected with S. mansoni, the characterization of this S. mansoni cyclophilin homologue may allow a better understanding of the schistosomicidal nature of cyclosporin A and lead to a novel strategy of therapy for schistosomiasis.

Cyclic AMP modifies the cellular distribution of connexin43 and induces a persistent increase in the junctional permeability of mouse mammary tumor cells.

Direct communication between cells via gap junctions is thought to be an important component of homeostasis and coordinated cellular responses to external signals. We investigated how the second messenger cAMP exerts its effects on junctional communication in a mouse mammary tumor cell line, MMT22. Junctional permeance was quantitatively assessed using dye microinjection and video microscopy. An increase of permeance was found after exposure to 8-bromo-cAMP, being detectable after 30 minutes of treatment and attaining a fourfold higher level of permeance by 24 hours. This elevated level was maintained with continuous exposure to 8-bromo-cAMP for seven days. The permeability change was accompanied by an increase in gap junctions as shown by freeze-fracture electron microscopy and by confocal microscopy using antibodies directed against the gap junction protein, connexin43. The amount of detergent-insoluble connexin43 also increased with 8-bromo-cAMP treatment, and most of the increase could be attributed to an increase of slower migrating (i.e. phosphorylated) species of connexin43. However, connexin43 mRNA and the total cellular content of connexin43 did not change over this period of exposure to 8-bromo-cAMP, as shown by densitometric analyses of northern and western blots. We conclude that 8-bromo-cAMP affects the distribution of connexin43 such that a greater proportion of the protein is utilized for channel formation. Since these changes were relatively slow to develop and persisted with prolonged exposure to 8-bromo-cAMP, it is possible that the junctional permeability of these mammary tumor cells is linked to the 'basal' level of cAMP, i.e. levels maintained by the cells in accordance with a particular cell state.

Analysis of multiple gap junction gene products in the rodent and human mammary gland.

The expression and localization of three different connexins (alpha 1, Cx43; beta 1, Cx32; and beta 2, Cx26) were analyzed in human, mouse, and rat mammary glands by PCR analysis of reverse-transcribed RNA (RT-PCR) and indirect immunohistochemistry. For the rodent mammary gland, the study included different physiological stages of development during nonpregnancy, pregnancy, lactation, and postweaning. RT-PCR amplification revealed a constitutive expression of RNA for alpha 1 connexin in all three species. In contrast, both beta 1 and beta 2 transcripts were expressed only during lactation in the rodent mammary gland. Specifically, immunohistochemistry showed that the expression of all three connexins was restricted to specific cell types, and it varied according to the physiological activity of the organ. In particular, alpha 1 antigen was detected only between myoepithelial cells, the contractile cells surrounding alveoli and ductal systems in the mammary gland, while beta 1 and beta 2 antigens were localized solely at the basolateral border of alveolar secretory cells. The level of beta 1 and beta 2 connexins was increased in the rodent mammary gland during lactation. No staining for either beta connexin was detected in human mammary gland or in that of nonpregnant, pregnant, or postweaning rodents. The inducible coexpression of beta 1 and beta 2 antigens in luminal cells of the lactating rodent suggests a possible role for these connexins in the coordination of the secretory epithelium.

Schistosoma mansoni: characterization of p50, an immunophilin.

A 1.4-kb cDNA clone encoding a 50-kDa homologue of p59, a heat shock binding immunophilin, has been isolated from a Schistosoma mansoni cercariae cDNA library. We have designated this clone S. mansoni p50 (Smp50). From the sequence comparison, we speculate that Smp50 is similar in structural organization to other p59 proteins. As with other p59s, Smp50 also shows homology to various FK binding proteins (FKBPs). The amino acids in human FKBP12 which are proposed to be important for FK506 interaction are conserved in the schistosome protein. We have expressed and purified the recombinant protein (rSmp50) from Escherichia coli. rSmp50 demonstrated peptidyl-prolyl cis-transisomerase (PPIase) activity, typical of immunophilins. Western blot analysis of extracts from S. mansoni adult worms probed with rabbit polyclonal antisera, generated against the recombinant polypeptide, has indicated the presence of Smp50 in the parasite. The antisera did not cross-react with proteins from E. coli or HeLa cell extracts. All of the p59s characterized to date have been from vertebrate species. Therefore, our finding represents the first identification of the protein in an invertebrate system.

Alternating chemotherapy regimens for patients with metastatic breast cancer. A pilot study based on tumor marker kinetics. Cancer and Leukemia Group B.

BACKGROUND: Chemotherapy is most effective when applied during the biologically active stage of tumor cells. According to the authors' previous tumor marker kinetic study, methotrexate plus 5-fluorouracil (MF) was found to yield either a cytolytic effect in an MF-sensitive tumor cell population or a cytostatic effect in an MF-resistant population. In the latter, the suppressive effect was transient and the biologic activity resumed in one week after MF administration. METHODS: Based on this marker kinetic study, an alternating chemotherapy program was designed to study its antitumor and side effects. Methotrexate (M) (200 mg/m2) and 5-fluorouracil (F) (500 mg/m2) were administered intravenously on day 1 followed 24 hours later by leucovorin (L) (10 mg/m2 orally every 6 hours for 6 doses). Cyclophosphamide (C) 300 (mg/m2), doxorubicin (A) (50 mg/m2), and vincristine (V) (1 mg/m2) were given on day 8. The MFL/CAV was given every 4 weeks. RESULTS: Forty-nine patients with metastatic breast cancer were enrolled; 41 were eligible. There were 5 complete and 23 partial remissions, producing a total response rate of 68%. In 15 patients with liver metastases, the response rate was 73% and the median survival 13.7 months, results superior to those previously reported for this subgroup of patients. Side effects were manageable. CONCLUSIONS: This regimen, which can be given safely in an outpatient setting, yielded encouraging response and survival rates in patients with visceral-dominant disease with poor prognoses.

Measurement of gap junctional communication by fluorescence activated cell sorting.

Cell-to-cell communication via gap junctions has played a fundamental role in the orderly development of multicellular organisms. Current methods for measuring this function apply mostly to homotypic cell populations. The newly introduced Fluorescence Activated Cell Sorting (FACS) method, albeit with some limitations, is simple, reliable, and quantitative in measuring the dye transfer via gap junctions in both homotypic and heterotypic cell populations. In the homotypic setting, the result in dye transfer from the FACS method is comparable to the scrape-loading and microinjection methods. Using this FACS method, we observed a decline of cell-to-cell communication in transformed and cancer cells. We also observed a differential degree of communication between two heterotypic cell populations depending on the direction of dye transfer.