Overcoming resistance to mitochondrial apoptosis by BZML‐induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells

Objectives

Our previous in vitro study showed that 5‐(3, 4, 5‐trimethoxybenzoyl)‐4‐methyl‐2‐(p‐tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti‐cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML‐treated A549 and A549/Taxol cells.

Methods

Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC‐1 staining, Annexin V‐FITC/PI double‐staining, a caspase‐9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity.

Results

Our data showed that BZML also exhibited desirable anti‐cancer activity against drug‐resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS‐mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non‐protective type of autophagy during BZML‐induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML‐induced MC in A549/Taxol cells.

Conclusions

Our data suggest that the anti‐apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML‐induced MC to overcome resistance to mitochondrial apoptosis.

     

Coroglaucigenin induces senescence and autophagy in colorectal cancer cells

Objectives

Coroglaucigenin (CGN), a natural product isolated from Calotropis gigantean by our research group, has been identified as a potential anti‐cancer agent. However, the molecular mechanisms involved remain poorly understood.

Materials and methods

Cell viability and cell proliferation were detected by MTT and BrdU assays. Flow cytometry, SA‐β‐gal assay, western blotting and immunofluorescence were performed to determine CGN‐induced apoptosis, senescence and autophagy. Western blotting, siRNA transfection and coimmunoprecipitation were carried out to investigate the mechanisms of CGN‐induced senescence and autophagy. The anti‐tumour activities of combination therapy with CGN and chloroquine were observed in mice tumour models.

Results

We demonstrated that CGN inhibits the proliferation of colorectal cancer cells both in vitro and in vivo. We showed that the inhibition of cell proliferation by CGN is independent of apoptosis, but is associated with cell‐cycle arrest and senescence in colorectal cancer cells. Notably, CGN induces protective autophagy that attenuates CGN‐mediated cell proliferation. Functional studies revealed that CGN disrupts the association of Hsp90 with both CDK4 and Akt, leading to CDK4 degradation and Akt dephosphorylation, eventually resulting in senescence and autophagy, respectively. Combination therapy with CGN and chloroquine resulted in enhanced anti‐tumour effects in vivo.

Conclusions

Our results demonstrate that CGN induces senescence and autophagy in colorectal cancer cells and indicate that combining it with an autophagy inhibitor may be a novel strategy suitable for CGN‐mediated anti‐cancer therapy.

     

Genista sessilifolia DC. extracts induce apoptosis across a range of cancer cell lines

Objectives

Restorative properties of medicinal plants such as Genista sessilifolia DC. have often been suggested to occur, in epidemiological studies. However, full characterization of effective principles responsible for this action has never previously been performed. Here, we have characterized G. sessilifolia's anti‐cancer effects and identified the chemical components involved in this anti‐tumour action.

Materials and methods

Cell cycle, apoptosis, necrosis, differentiation analyses, high‐performance liquid chromatography, western blotting, RNA extraction, real‐time PCR and primers have all been observed/used in the study.

Results

We report that G. sessilifolia methanol extract has anti‐cancer activity on solid and haematological cancer cells. G. sessilifolia extract's anti‐proliferative action is closely bound to induction of apoptosis, whereas differentiation is only weakly modulated. Analysis of G. sessilifolia extract, by high‐performance liquid chromatography, identifies fraction 18–22 as the pertinent component for induction of apoptosis, whereas fractions 11–13 and 27–30 both seem to contribute to differentiation. G. sessilifolia extract induces apoptosis mediated by caspase activation and p21, Rb, p53, Bcl2‐associated agonist of cell death (BAD), tumour necrosis factor receptor super‐family, member 10 (TRAIL) overexpression and death receptor 5 (DR5). Accordingly, fraction 18–22 inducing apoptosis was able to induce TRAIL.

Conclusions

Our results indicate that G. sessilifolia extract and its fraction 18–22 containing genistin and isoprunetin, were able to induce anti‐cancer effects supporting the hypothesis of a pro‐apoptotic intrinsic content of this natural medicinal plant.

     

A novel inhibitor of ADAM17 sensitizes colorectal cancer cells to 5‐Fluorouracil by reversing Notch and epithelial‐mesenchymal transition in vitro and in vivo

Objectives

Colorectal cancer is one of the most common malignancies both in men and women. Owing to metastasis and resistance, the prognosis of colorectal cancerCRC patients remains extremely poor with chemotherapy. A disintegrin and metalloproteinase 17 (ADAM17) induces the activation of Notch pathway and contributes to the chemoresistance. This study aimed to discover a novel ADAM17 inhibitor and investigate the chemosensitization effect.

Materials and methods

Pharmacophore model, western blot and enzymatic assay were used to discover ZLDI‐8. Cell proliferation was determined by MTT and colony formation assay. Cell migratory and invasive ability were determined by wound healing scratch and transwell assay. Immunofluorescence images and western blot analysed the expression of Notch or epithelial‐mesenchymal transition (EMT) pathway markers. Xenografts were employed to evaluate the chemosensitization effect of ZLDI‐8 in vivo.

Results

We found that ZLDI‐8 cell‐specifically inhibited the proliferation of CRC, and this effect was due to abrogation of ADAM17 and Notch pathway. Meanwhile, we reported for the first time that ZLDI‐8 synergistically improved the anti‐tumour and anti‐metastasis activity of 5‐fluorouracil or irinotecan by reversing Notch and EMT pathways. Interestingly, in vivo studies further demonstrated that ZLDI‐8 promoted the anti‐tumour effect of 5‐fluorouracil through Notch and EMT reversal.

Conclusions

A novel ADAM17 inhibitor ZLDI‐8 may be a potential chemosensitizer which sensitized CRC cells to 5‐fluorouracil or irinotecan by reversing Notch and EMT pathways.

     

Tocotrienols promote apoptosis in human breast cancer cells by inducing poly(ADP‐ribose) polymerase cleavage and inhibiting nuclear factor kappa‐B activity

Objectives

Tocotrienols and tocopherols are members of the vitamin E family, with similar structures; however, only tocotrienols have been reported to achieve potent anti‐cancer effects. The study described here has evaluated anti‐cancer activity of vitamin E to elucidate mechanisms of cell death, using human breast cancer cells.

Materials and methods

Anti‐cancer activity of a tocotrienol‐rich fraction (TRF) and a tocotrienol‐enriched fraction (TEF) isolated from palm oil, as well as pure vitamin E analogues (α‐tocopherol, α?, δ? and γ?tocotrienols) were studied using highly aggressive triple negative MDA‐MB‐231 cells and oestrogen‐dependent MCF‐7 cells, both of human breast cancer cell lines. Cell population growth was evaluated using a Coulter particle counter. Cell death mechanism, poly(ADP‐ribose) polymerase cleavage and levels of NF‐κB were determined using commercial ELISA kits.

Results

Tocotrienols exerted potent anti‐proliferative effects on both types of cell by inducing apoptosis, the underlying mechanism of cell death being ascertained using respective IC₅₀ concentrations of all test compounds. There was marked induction of apoptosis in both cell lines by tocotrienols compared to treatment with Paclitaxel, which was used as positive control. This activity was found to be associated with cleavage of poly(ADP‐ribose) polymerase (a DNA repair protein), demonstrating involvement of the apoptotic cell death signalling pathway. Tocotrienols also inhibited expression of nuclear factor kappa‐B (NF‐κB), which in turn can increase sensitivity of cancer cells to apoptosis.

Conclusion

Tocotrienols induced anti‐proliferative and apoptotic effects in association with DNA fragmentation, poly(ADP‐ribose) polymerase cleavage and NF‐κB inhibition in the two human breast cancer cell lines.

     

The inhibitory effects of capillarisin on cell proliferation and invasion of prostate carcinoma cells

Objectives

Capillarisin (Cap), an active component of Artemisia capillaris root extracts, is characterized by its anti‐inflammatory, anti‐oxidant and anti‐cancer properties. Nevertheless, the functions of Cap in prostate cancer have not been fully explored. We evaluated the potential actions of Cap on the cell proliferation, migration and invasion of prostate carcinoma cells.

Materials and methods

Cell proliferation and cell cycle distribution were measured by water‐soluble tetrazolium‐1 and flow cytometry assays. The expression of cyclins, p21, p27, survivin, matrix metallopeptidase (MMP2 and MMP9) were assessed by immunoblotting assays. Effects of Cap on invasion and migration were determined by wound closure and matrigel transmigration assays. The constitutive and interlukin‐6 (IL‐6)‐inducible STAT3 activation of prostate carcinoma cells were determined by immunoblotting and reporter assays.

Results

Capillarisin inhibited androgen‐independent DU145 and androgen‐dependent LNCaP cell growth through the induction of cell cycle arrest at the G0/G1 phase by upregulating p21 and p27 while downregulating expression of cyclin D1, cyclin A and cyclin B. Cap decreased protein expression of survivin, MMP‐2, and MMP‐9 and therefore blocked the migration and invasion of DU145 cells. Cap suppressed constitutive and IL‐6‐inducible STAT3 activation in DU145 and LNCaP cells.

Conclusions

Our data indicate that Cap blocked cell growth by modulation of p21, p27 and cyclins. The inhibitory effects of Cap on survivin, MMP‐2, MMP‐9 and STAT3 activation may account for the suppression of invasion in prostate carcinoma cells. Our data suggest that Cap might be a therapeutic agent in treating advanced prostate cancer with constitutive STAT3 or IL‐6‐inducible STAT3 activation.

     

Mesenchymal stem cell transfusion for desensitization of positive lymphocyte cross‐match before kidney transplantation: outcome of 3 cases

Objectives

Donor specific antibodies (DSA) and a positive cross‐match are contraindications for kidney transplantation. Trials of allograft transplantation across the HLA barrier have employed desensitization strategies, including the use of plasmapheresis, intravenous immunoglobulins, anti‐B‐cell monoclonal antibodies and splenectomy, associated with high‐intensity immunosuppressive regimens. Our case 1 report suffered from repeatedly positive lymphocyte cross match after 1st renal transplantation. Graft nephrectomy could not correct the state of sensitization. Splenectomy was done in a trial to get rid of the antibody producing clone. Furthermore plasmapheresis with low dose IVIG could not as well revert the state of sensitization for the patient.

Material and methods

About 50 millions donor specific MSCs were injected to the patient.

Results

MSCs transfusion proved to be the only procedure which could achieve successful desensitization before performing the second transplantation owing to their immunosuppressive properties.

Conclusion

This case indicates that DS‐MSCs is a potential option for anti‐HLA desensitization. In cases 2 and 3 IV DS‐MSCs transfusion was selected from the start as a successful line of treatment for pre renal transplantation desensitization to save other unnecessary lines of treatment that were tried in case 1.

     

Low level laser therapy induces increased viability and proliferation in isolated cancer cells

Objectives

Low level laser therapy (LLLT), which stimulates natural biological processes in the application region, is frequently used in dental treatments. The aim of our study was to evaluate the effects of LLLT which could activate precancerous cells or increase existing cancerous tissue in case of clinically undetectable situations.

Materials and methods

Saos‐2 osteoblast‐like osteosarcoma cells and A549 human lung carcinoma cells were used. Twenty‐four hours after preparation of cell culture plates, laser irradiation was performed 1, 2 and 3 times according to the test groups using Nd:YAG laser with the power output 0.5, 1, 2 and 3 W. Cell proliferation analysis was performed by MTT assay at the 24th hour following the last laser applications.

Results

Generally, it was observed that the proliferation rates increased as the number of applications increased, when compared to the controls, especially in those cases in which the irradiation was performed 2 or 3 times more.

Conclusion

The findings of this study have led to the conclusion that LLLT increases cancer cell proliferation, depending on the power output level of the laser and the number of applications. In addition to the proliferation and mitotic activity of the cancer tissue cells, we concluded that LLLT, which is frequently used in dental practice, could activate precancerous cells or increase existing cancerous tissue.

     

Curcumin enhances the anti‐cancer effects of Paris Saponin II in lung cancer cells

Objectives

To investigate the synergistic mechanisms of Paris Saponin II (PSII) and Curcumin (CUR) in lung cancer.

Materials and Methods

The combination changed the cellular uptake of CUR and PSII, apoptosis, cell cycle arrest and cytokine levels were analysed on different lung cancer cells.

Results

The combination displayed a synergistic anti‐cancer effect through promoting the cellular uptake of CUR on different lung cancer cells. Hoechst H33258 staining and FACS assay indicated that the combination of PSII and CUR induced cell cycle arrest and apoptosis. Western blot and cytokine antibody microarray suggested that the combination activated death receptors such as DR6, CD40/CD40L, FasL and TNF‐α to induce cancer cells apoptosis, and up‐regulated IGFBP‐1 leading to inhibition of PI3K/Akt pathway and increase of p21 and p27, which therefore induced a G2 phase arrest in NCI‐H446 cells. Meanwhile, the combination suppressed PCNA and NF‐κB pathway in 4 kinds of lung cancer cells. They activated the phosphorylation of p38 and JNK, and inhibited PI3K in NCI‐H460 and NCI‐H446 cells, enhanced the phosphorylation of JNK in NCI‐H1299 cells, and increased the phosphorylation of p38 and ERK, and suppressed PI3K in NCI‐H520 cells.

Conclusions

PSII combined with CUR had a synergistic anti‐cancer effect on lung cancer cells. These findings provided a rationale for using the combination of curcumin and PSII in the treatment of lung cancer in future.

     

Fluoxetine induces autophagic cell death via eEF2K‐AMPK‐mTOR‐ULK complex axis in triple negative breast cancer

Objectives

Triple negative breast cancer (TNBC) is a complex and intrinsically aggressive tumour with poor prognosis, and the discovery of targeted small‐molecule drugs for TNBC treatment still remains in its infancy. In this study, we aimed to discover a small‐molecule agent for TNBC treatment and illuminate its potential mechanisms.

Materials and methods

Cell viability was detected by using methylthiazoltetrazolium (MTT) assay. Electron microscopy, GFP‐LC3 transfection, monodansylcadaverine staining and apoptosis assay were performed to determine Fluoxetine‐induced autophagy and apoptosis. Western blotting and siRNA transfection were carried out to investigate the mechanisms of Fluoxetine‐induced autophagy. iTRAQ‐based proteomics analysis was used to explore the underlying mechanisms.

Results

We have demonstrated that Fluoxetine had remarkable anti‐proliferative activities and induced autophagic cell death in MDA‐MB‐231 and MDA‐MB‐436 cells. The mechanism for Fluoxetine‐induced autophagic cell death was associated with inhibition of eEF2K and activation of AMPK‐mTOR‐ULK complex axis. Further iTRAQ‐based proteomics and network analyses revealed that Fluoxetine‐induced mechanism was involved in BIRC6, BNIP1, SNAP29 and Bif‐1.

Conclusions

These results demonstrate that Fluoxetine induces apoptosis and autophagic cell death in TNBC, which will hold a promise for the future TNBC therapy.

     

Effects of simvastatin and rosuvastatin on RAS protein,matrix metalloproteinases and NF‐κB in lung cancer and in normal pulmonary tissues

Objectives

In this study, we have evaluated effects of 24‐hour treatments with simvastatin or rosuvastatin on RAS protein, NF‐κB and MMP expression in LC tissues obtained from 12 patients undergoing thoracic surgery.

Materials and methods

Normal and lung tumour tissues obtained from each sample were exposed to simvastatin (2.5–30 μm ) or rosuvastatin (1.25–30 μm ) and western blot analysis was then performed.

Results

We documented increased expression of proteins, MMP‐2, MMP‐9 and NF‐κB‐p65 in LC tissues, with respect to normal tissues (P < 0.01). In the malignant tissues, simvastatin and rosuvastatin significantly (P < 0.01) and dose‐dependently reduced RAS protein, MMP‐2/9 and NF‐κB‐p65 expression.

Conclusions

In conclusion, our results suggest that simvastatin and rosuvastatin could play a role in LC treatment by modulation of RAS protein, MMP‐2/9 and NF‐κB‐p65.

     

Low‐intensity pulsed ultrasound induced enhanced adipogenesis of adipose‐derived stem cells

Objective

The aim of this study was to investigate effects of low‐intensity pulsed ultrasound (LIPUS) on differentiation of adipose‐derived stem cells (ASCs), in vitro.

Materials and methods

Murine ASCs were treated with LIPUS for either three or five days, immediately after adipogenic induction, or delayed for 2 days. Expression of adipogenic genes PPAR‐γ1, and APN, was examined by real‐time PCR. Immunofluorescence (IF) staining was performed to test for PPAR‐γ at the protein level.

Results

Our data revealed that specific patterns of LIPUS up‐regulated levels of both PPAR‐γ1 and APN mRNA, and PPAR‐γ protein.

Conclusions

In culture medium containing adipogenic reagents, LIPUS enhanced ASC adipogenesis.

     

In vitro cytotoxicity of Gymnema montanum in human leukaemia HL‐60 cells; induction of apoptosis by mitochondrial membrane potential collapse

Objectives

Gymnema montanum Hook, an Indian Ayurvedic medicinal plant, is used traditionally to treat a variety of ailments. Here, we report anti‐cancer effects and molecular mechanisms of ethanolic extract of G. montanum (GLEt) on human leukaemia HL‐60 cells, compared to peripheral blood mononuclear cells.

Materials and methods

HL‐60 cells were treated with different concentrations of GLEt (10–50 μg/ml) and cytotoxicity was assessed by MTT assay. Levels of lipid peroxidation, antioxidants, mitochondrial membrane potential and caspase‐3 were measured. Further, apoptosis was studied using annexin‐V staining and the cell cycle was analyzed by flow cytometry.

Results

GLEt had a potent cytotoxic effect on HL‐60 cells (IC₅₀‐20 μg/ml), yet was not toxic to normal peripheral blood mononuclear cells. Exposure of HL‐60 cells to GLEt led to elevated levels of malonaldehyde formation, but to reduced glutathione, superoxide dismutase, catalase and glutathione peroxidase activities (P < 0.05). Induction of apoptosis was confirmed by observing annexin‐V positive cells, associated with loss of mitochondrial membrane potential. Cell cycle arrest at G0/G1 was observed in GLEt‐treated HL‐60 cells, indicating its potential at inducing their apoptosis.

Conclusions

Findings of the present study suggest that G. montanum induced apoptosis in the human leukaemic cancer cells, mediated by collapse of mitochondrial membrane potential, generation of reactive oxygen species and depletion of intracellular antioxidant potential.

     

2‐(2‐nitrobenzylidene) indolin‐3‐one compound inhibits transmembrane prostate androgen‐induced protein (TMEPAI) expression and cancer cell proliferation

Objectives

The transmembrane prostate androgen‐induced protein (TMEPAI) is aberrantly expressed in many cancer and plays a crucial role in tumourigenesis, which makes it a potential cancer therapeutic target for drug discovery.

Materials and methods

Here, we employed a firefly luciferase reporter driven by the TMEPAI gene promoter to screen for compound capable of inhibiting the expression of TMEPAI, and the effects of TMEPAI inhibitor on cancer cell proliferation were evaluated using the colony formation assay, cell cycle analysis, Ki‐67 immunofluorescence assay and EdU incorporation assay.

Results

2‐(2‐nitrobenzylidene) indolin‐3‐one (JHY‐A007‐50) was identified and shown to effectively inhibit the TMEPAI promoter activity. Further studies revealed that JHY‐A007‐50 specifically inhibited the expression of TMEPAI at both the mRNA and protein levels. Moreover, we found that JHY‐A007‐50 could inhibit cell proliferation and induce cell cycle arrest at the G1 phase. Our results showed that overexpression of TMEPAI decreased the inhibitory effects of JHY‐A007‐50 on cancer cell proliferation, and JHY‐A007‐50 did not affect the cell viability of HeLa cells knocked down of TMEPAI.

Conclusions

Taken together, these results suggest that compound JHY‐A007‐50 mediates the downregulation of TMEPAI expression and inhibits cell proliferation in cancer cells.

     

Hypericum perforatum L. subsp. perforatum induces inhibition of free radicals and enhanced phototoxicity in human melanoma cells under ultraviolet light

Objectives

Our interest continues in discovering phytocomplexes from medicinal plants with phototoxic activity against human melanoma cells; thus the aim of the present study was to assess antioxidant, anti‐inflammatory and phototoxic activity of Hypericum perforatum L. subsp. perforatum, and relate these properties to the plant's chemical composition.

Materials and methods

Components of H. perforatum subsp. perforatum were extracted by hydroalcoholic solution and chemical profiles of preparations (HyTE‐3) performed by HPTLC. Linoleic acid peroxidation and DPPH tests were used to assess antioxidant activity, while MTT assay allowed evaluation of anti‐proliferative activity with respect to A375 human melanoma cells after irradiation with UVA dose, 1.8 J/cm². Inhibition of nitric oxide production of macrophages was also investigated.

Results

HyTE‐3 indicated better antioxidant activity with β‐carotene bleaching test in comparison to DPPH assay (IC₅₀ = 0.89 μg/ml); significant phototoxicity in A375 cells at 78 μg/ml concentration resulted in cell destruction of 50%. HyTE‐3 caused significant dose‐related inhibition of nitric oxide production in murine monocytic macrophage cell line RAW 264.7 with IC₅₀ value of 342 μg/ml.

Conclusions

The H. perforatum subsp. perforatum‐derived product was able to suppress proliferation of human malignant melanoma A375 cells; extract together with UVA irradiation enhanced phototoxicity. This biological activity of antioxidant effects was combined with inhibition of nitric oxide production.

     

Identification of novel caspase/autophagy‐related gene switch to cell fate decisions in breast cancers

Objectives

Caspases, a family of cysteine proteases with unique substrate specificities, contribute to apoptosis, whereas autophagy‐related genes (ATGs) regulate cytoprotective autophagy or autophagic cell death in cancer. Accumulating evidence has recently revealed underlying mechanisms of apoptosis and autophagy; however, their intricate relationships still remain to be clarified. Identification of caspase/ATG switches between apoptosis and autophagy may address this problem.

Materials and methods

Identification of caspase/ATG switches was carried out using a series of elegant systems biology & bioinformatics approaches, such as network construction, hub protein identification, microarray analyses, targeted microRNA prediction and molecular docking.

Results

We computationally constructed the global human network from several online databases and further modified it into the basic caspase/ATG network. On the basis of apoptotic or autophagic gene differential expressions, we identified three molecular switches [including androgen receptor, serine/threonine‐protein kinase PAK‐1 (PAK‐1) and mitogen‐activated protein kinase‐3 (MAPK‐3)] between certain caspases and ATGs in human breast carcinoma MCF‐7 cells. Subsequently, we identified microRNAs (miRNAs) able to target androgen receptor, PAK‐1 and MAPK‐3, respectively. Ultimately, we screened a range of small molecule compounds from DrugBank, able to target the three above‐mentioned molecular switches in breast cancer cells.

Conclusions

We have systematically identified novel caspase/ATG switches involved in miRNA regulation, and predicted targeted anti‐cancer drugs. These findings may uncover intricate relationships between apoptosis and autophagy and thus provide further new clues towards possible cancer drug discovery.

     

Elucidation of proliferative capability of mononuclear tetraploid cells,emerging spontaneously from diploid cells,using image cytometry and fluorescence in situ hybridization

Objectives

Proliferation of tetraploid cells (TCs) emerging from diploid cells is considered to be a critical event toward tumourigenesis, or cancer progression. Recently, several studies have reported that binuclear TCs emerging from normal cells are capable of mitosis, however, it has not been confirmed directly whether mononuclear TCs emerging from normal cells could proliferate, even cancer cells. The aim of this study is to detect mononuclear TCs in vitro, spontaneously emerging from diploid cells and to elucidate their proliferative capability directly. For this purpose, we have developed a novel method.

Materials and methods

In this study, two completely disomic cell lines were used, TIG‐7, a fibroblast cell line and CAL‐51, a breast cancer cell line. Cells were cultured on microscope slides and their DNA content was determined using an image cytometer. On the same slides, chromosome numbers were scored using centromere fluorescence in situ hybridization (FISH). For evaluating proliferative capability of TCs, bromodeoxyuridine (BrdUrd) incorporation and colony‐forming ability were examined.

Results

Using our method, spontaneous emergence of mononuclear TCs was detected in both TIG‐7 and CAL‐51. Colonies of TIG‐7 TCs were not observed, but were observed of CAL‐51 TCs.

Conclusions

Our method enables detection of mononuclear TCs and elucidation of their proliferative capability, directly; this evidence reveals that mononuclear TIG‐7 TCs do not proliferate but that mononuclear CAL‐51 TCs are able to.