AI-based Apoptosis Cell Classification Using Phase-contrast Images of K562 Cells.

BACKGROUND/AIM: This study aimed to automate the classification of cells, particularly in identifying apoptosis, using artificial intelligence (AI) in conjunction with phase-contrast microscopy. The objective was to reduce reliance on manual observation, which is often time-consuming and subject to human error. MATERIALS AND METHODS: K562 cells were used as a model system and apoptosis was induced following administration of gamma-secretase inhibitors. Fluorescence staining was applied to detect DNA fragmentation and caspase activity. Cell images were obtained using both phase-contrast and fluorescence microscopy. Two AI models, Lobe(R) and a server-based ResNet50, were trained using these images and evaluated using F-values through five-fold cross-validation. RESULTS: Both AI models demonstrated effectively categorized individual cells into three groups: caspase-negative/no DNA fragmentation, caspase-positive/no DNA fragmentation, and caspase-positive/DNA fragmentation. Notably, the AI models' ability to differentiate cells relied on subtle variations in phase-contrast images, potentially linked to changes in refractive indices during apoptosis progression. Both AI models exhibited high accuracy, with the server-based ResNet50 model showing improved performance through repeated training. CONCLUSION: This study demonstrates the potential of AI-assisted phase-contrast microscopy as a powerful tool for automating cell classification, especially in the context of apoptosis research and the discovery of anticancer substances. By reducing the need for manual labor and enhancing classification accuracy, this approach holds promise for expediting high-throughput cell screening, significantly contributing to advancements in medical diagnostics and drug development.

Hypoxia Up-regulates HIF Expression While Suppressing Cell Growth and NOTCH Activity in Leukaemia Cells.

AIM: To examine the influence of hypoxia on the in vitro growth of leukaemia cells and the activity of signalling proteins to better understand the pathophysiology of leukaemia cells in human bone marrow. MATERIALS AND METHODS: Six human leukaemia cell lines were cultured under normoxic or hypoxic conditions. Cell growth, recovery of clonogenic cells, and the expression and activation of various signalling proteins were examined. RESULTS: Hypoxia suppressed cell growth and the recovery of clonogenic cells. Moreover, hypoxia up-regulated hypoxia-inducible factor (HIF) 1α and HIF2α expression while suppressing the expression and activation of NOTCH1, mechanistic target of rapamycin kinase (mTOR) activation, and nuclear factor-kappa B (NF-κB) phosphorylation. CONCLUSION: We found that hypoxia up-regulated HIF expression while it suppressed the self-renewal capacity of leukaemia cells, NOTCH activity, and expression of its down-stream signalling molecules, which differs from previous reports mentioning that HIF activates NOTCH signalling. Our findings serve to further elucidate the in vivo pathophysiology of leukaemia cells.

GLI1 and CTNNB1 Knockdown Activates NOTCH and mTOR Signalling in NB4 Myeloid Leukaemia Cells.

BACKGROUND: Hedgehog (HH), WNT, NOTCH, and mechanistic target of rapamycin (mTOR) signalling pathways are known to regulate the progression of cancer; however, their interaction in leukaemia cells is not fully clarified. MATERIALS AND METHODS: Myeloid and T-lymphoblastic leukaemia cell lines (NB4, THP-1, Jurkat, and DND-41) were transfected with small interfering RNAs targeting the glioma-associated oncogene homolog 1 (GLI1) and catenin beta-1 (CTNNB1) genes involved in the regulation of HH and WNT pathways, respectively, and we examined cell proliferation and gene expression. RESULTS: The knockdown of GLI1 and CTNNB1 did not significantly affect proliferation of any cell line; however, it up-regulated the expression of NOTCH1, cleaved NOTCH1 fragment, and phosphorylated mTOR in NB4 cells, but not in the other cell lines. CONCLUSION: Our data suggest that HH and WNT act upstream of NOTCH and mTOR pathways and negatively regulate them in myeloid NB4 cells. Further studies are required to determine the biological significance of this signalling crosstalk in leukaemia.

Hedgehog Stimulation Suppresses Clonogenicity and Activates NOTCH Signalling in T-lymphoblastic Leukaemia Jurkat Cells.

BACKGROUND/AIM: Hedgehog (HH) and NOTCH pathways are involved in the regulation of cancer stem cells and haematopoietic malignancies. However, the effects of HH stimulation on cell growth and NOTCH signalling in acute T-lymphoblastic leukaemia (T-ALL) cells have not been elucidated. MATERIALS AND METHODS: Two T-ALL cell lines, Jurkat and KOPT-K1 harbouring activating NOTCH1 mutations, were cultured with recombinant Sonic (S) HH and analysed for proliferation, colony formation, and expression of NOTCH-regulated genes and proteins. RESULTS: SHH stimulation did not affect cell growth but suppressed colony formation, increased the levels of cleaved NOTCH1 fragment characteristic for NOTCH1 activation, and upregulated mRNA expression of HES1, while decreasing that of MYC in Jurkat cells. However, no such effects were observed in KOPT-K1 cells. CONCLUSION: Our results indicate that SHH stimulation activates NOTCH signalling in Jurkat cells, thus disclosing a novel relationship between HH and NOTCH pathways.

NOTCH knockdown affects the proliferation and mTOR signaling of leukemia cells.

AIM: The effects of small interfering RNA (siRNA)-mediated knockdown of NOTCH1 and NOTCH2 on cell proliferation and downstream signaling pathways in leukemia cells were examined. MATERIALS AND METHODS: Two T-lymphoblastic leukemia (T-ALL) cell lines and two acute myeloblastic leukemia (AML) cell lines were transfected with siRNAs targeting NOTCH1 and NOTCH2. The effects of knockdown on cell proliferation and protein expression were examined by colorimetric WST-8 assay and immunoblotting, respectively. RESULTS: In T-ALL cell lines, NOTCH1 knockdown as well as NOTCH2 knockdown suppressed cell proliferation and induced apoptosis. v-Myc avian myelocytomatosis viral oncogene homolog (MYC) protein expression was down-regulated in NOTCH1-knockdown cells but not affected in NOTCH2-knockdown cells. In AML cell lines, cell proliferation was not significantly affected by NOTCH siRNAs. NOTCH2 knockdown increased the level of cleaved NOTCH1 fragment without increasing NOTCH1 expression. NOTCH knockdown reduced the level of mechanistic target of rapamycin (mTOR) protein in the monoblastic leukemia cell line THP-1. Contrastingly, NOTCH activation by NOTCH ligand stimulation increased the expression of mTOR in THP-1 cells. CONCLUSION: These novel findings on NOTCH signaling may contribute to the development of effective NOTCH-targeted therapies against leukemia.

Effects of the HIF1 inhibitor, echinomycin, on growth and NOTCH signalling in leukaemia cells.

AIM: To examine the effects of echinomycin, a compound that inhibits DNA-binding activity of hypoxia-inducible factor-1 (HIF1), on leukaemia cell growth. MATERIALS AND METHODS: Three acute myeloid leukaemia cell lines and three T-lymphoblastic leukaemia cell lines were cultured with echinomycin. Cell growth, mRNA and protein expression levels were examined by WST-1 assay, reverse-transcription polymerase chain reaction and immunoblotting, respectively. RESULTS: HIF1α protein was expressed in all cell lines under normoxia. Treatment with echinomycin suppressed cell growth and induced apoptosis in association with decreased mRNA expression of HIF1 targets, glucose transporter-1 (GLUT1) and B-cell CLL/lymphoma-2 (BCL2). Echinomycin also suppressed the protein expression of NOTCH1, cleaved NOTCH1, v-myc myelocytomatosis viral oncogene homolog (MYC), v-akt murine thymoma viral oncogene homolog-1 (AKT), phosphorylated AKT, mechanistic target of rapamycin (mTOR), and phosphorylated mTOR and increased that of cleaved caspase-3 in some cell lines. CONCLUSION: Echinomycin suppresses leukaemia cell growth in association with reduced NOTCH1 expression. This is the first report to show that HIF inhibitor treatment suppresses NOTCH1 signalling. HIF inhibitors could be novel candidates for a molecular-targeted therapy against leukaemia.

Comparative effects of PP242 and rapamycin on mTOR signalling and NOTCH signalling in leukemia cells.

AIM: PP242 is a compound which inhibits both mammalian target of rapamycin complex-1 (mTORC1) and mTORC2. We examined the effects of PP242 and rapamycin on mTOR signalling and evaluated potential crosstalk with the NOTCH signalling in eight leukemia cell lines. MATERIALS AND METHODS: We examined the effects of treatment with these inhibitors on cell growth and protein expression. RESULTS: PP242 suppressed growth more potently than did rapamycin. In two cell lines poorly sensitive to PP242, PP242 failed to inhibit v-akt murine thymoma viral oncogene homolog (AKT) phosphorylation. Suppression of mTOR phosphorylation was weaker in myeloid cell lines. Rapamycin induced eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) hyperphosphorylation in three cell lines. Phosphorylation of both isoforms (p70 and p85) of S6 kinase (S6K) was suppressed in three cell lines; only p70 was suppressed in the others. NOTCH1 expression and activation were up-regulated by PP242 in one cell line but down-regulated in another. CONCLUSION: PP242 is a candidate for molecular-targeted leukemia therapy, although its effects must be evaluated on a case-by-case basis. Crosstalk was found between the mTOR and NOTCH signalling pathways.

Advantages of the quenching probe method over other PCR-based methods for detection of the JAK2 V617F mutation.

The detection of a V617F mutation (G to T exchange at nucleotide 1,849) in the JAK2 gene is crucial for the diagnosis of myeloproliferative neoplasms (MPN) such as polycythemia vera. Although sequence analysis is the standard method for detection, it is not suitable for clinical examinations due to the requirement of expensive equipment. In this study, we evaluated the efficiencies of four PCR-based methods to detect JAK2 V617F: allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), high-resolution melting analysis (HRM) and the quenching probe method (QP). The HEL cell line, which harbors a homozygous JAK2 V617F mutation, as well as bone marrow samples from 16 MPN patients and normal control samples, were used in this assessment. The sensitivity of the detection limit of all four methods was also examined using samples of HEL cells mixed in a variety of ratios with cells containing wild-type JAK2. The results of all four methods were found to be concordant. AS-PCR was shown to be the most sensitive; however, it produced false positive results. Although PCR-RFLP demonstrated high specificity, it was time consuming. By contrast, results were obtained using HRM and QP in only 2 h. It was easier to recognize the curves derived from the mutant allele obtained using QP. QP is also suitable for the rough estimation of allele burden. JAK2 V617F assays are mainly used for diagnosis at presentation in clinical settings. We therefore conclude that in situations where high sensitivity is not required, QP is the preferable method for the detection of JAK2 V617F. To the best of our knowledge, this is the first report to demonstrate the efficiency of the QP method for the detection of JAK2 V617F using a standard thermal cycler.

Effect of BMP4 on the growth and clonogenicity of human leukemia and lymphoma cells.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) signaling is involved in the maintenance of hematopoietic stem cells. However, the effects of BMP4 on leukemia and lymphoma cells are unknown. MATERIALS AND METHODS: The effects of recombinant BMP4 on the in vitro growth of 12 leukemia and lymphoma cell lines were examined. RESULTS: BMP4 treatment promoted the short-term growth of three cell lines and suppressed the growth of one. Induction of differentiation was not observed. BMP4 treatment suppressed the clonogenicity of four out of the six examined cell lines. BMP4 treatment promoted the growth of Jurkat cells but suppressed their ability to form colonies. BMP4 treatment up-regulated the phosphorylation of SMAD1/5/8 complex, indicating that BMP4 mediated signal transduction in the cells. CONCLUSION: BMP4 suppressed the clonogenicity of selected leukemia and lymphoma cell lines. The regulation of BMP4 signaling may be a useful therapeutic approach for leukemia and lymphoma, if appropriate cases are selected.

Promotion of the self-renewal capacity of human leukemia cells by sonic hedgehog protein.

BACKGROUND: Hedgehog (Hh) signaling is involved in cancer cell growth. However, the effects of Hh stimulation on leukemia cells are unknown. MATERIALS AND METHODS: The effects of recombinant sonic Hedgehog (Shh) protein on the in vitro growth of one B-lymphoma and four myeloid leukemia cell lines were examined. RESULTS: Shh stimulation had no significant effect on the short-term growth of whole cell populations in any of the five cell lines. However, Shh promoted clonogenic cell recovery after suspension culture, suggesting promotion of leukemia stem or progenitor cell amplification in three cell lines. The lack of Hh receptors in one cell line and endogenous Shh expression in another were possible reasons for the lack of effects of Shh in these cases. CONCLUSION: These results suggest that Shh stimulation promotes the self-renewal capacity of leukemia stem cells in some cell lines. Inhibition of Hh signaling could represent a novel therapeutic approach in leukemia.

Effects of combination of notch inhibitor plus hedgehog inhibitor or Wnt inhibitor on growth of leukemia cells.

BACKGROUND: The Notch inhibitors, γ-secretase inhibitors (GSIs), are promising candidates for molecular targeted therapy against leukemia. However, they show only limited effectiveness. We thought that the efficacy of GSIs might be improved by their combination with Hedgehog inhibitors and Wnt inhibitors because these signaling pathways are also important for the growth of leukemia cells. MATERIALS AND METHODS: The effects of the combination of GSI-XXI plus the Hedgehog inhibitor, cyclopamine (Cy), or the Wnt inhibitor, quercetin (Qu), on the in vitro cell growth, colony formation and Notch1 protein expression of three T-cell acute lymphoblastic leukemia (T-ALL) cell lines with NOTCH1 mutations and three acute myeloid leukemia cell lines were examined. RESULTS: The addition of Cy or Qu to GSI suppressed the growth of DND-41 T-ALL cells additively or synergistically, respectively. Interestingly, Cy treatment and Qu treatment reduced Notch1 protein and its active fragment in DND-41 cells, which suggests a relationship between Notch signaling and Hedgehog or Wnt signaling. The addition of Cy or Qu to GSI promoted the decrease of Notch1 activation and expression. CONCLUSION: The anti-leukemic effects of a GSI could be promoted by its combination with Cy or Qu, in appropriate cases selected by in vitro drug sensitivity test.

Gamma-secretase inhibitors induce erythroid differentiation in erythroid leukemia cell lines.

BACKGROUND: Notch signaling regulates the fate of hematopoietic stem cells and leukemia cells. However, the role of Notch in erythroid differentiation remains unclear. MATERIALS AND METHODS: We examined the effects of three γ-secretase inhibitors (GSI-IX, GSI-XII and GSI-XXI) that inhibit Notch signaling on the in vitro growth and differentiation of HEL and AA erythroid leukemia cell lines. RESULTS: GSI treatment induced morphologic erythroid differentiation and promoted hemoglobin production. GSI treatment suppressed short-term growth and colony formation, while treatment with GSI-XXI promoted the growth of AA cells. The degree of differentiation induced by each GSI roughly correlated with the reduction in HES1 mRNA expression. CONCLUSION: GSIs have potential uses in differentiation induction therapy for erythroid leukemia in the future. Before clinical use, in vitro sensitivity tests should be performed because the effects of GSIs are diverse depending upon the combination of leukemia cells and GSIs.

Effects of gamma-secretase inhibitors on the growth of leukemia cells.

BACKGROUND: Notch activation is involved in the growth of leukemia cells. gamma-Secretase inhibitors (GSIs), which block Notch activation, may be candidates for molecular target therapy against leukemia. MATERIALS AND METHODS: The effects of three kinds of GSIs: GSI-IX, GSI-XII and GSI-XXI, on the in vitro growth of various leukemia cell lines were examined. RESULTS: The effects of GSI were diverse depending upon the combination of cells and GSI. GSI treatment suppressed the growth of most of the cell lines examined. Conversely, the growth of some cell lines were promoted by GSI-XXI. GSI-XXI treatment reduced the amount of cleaved Notch1 protein and HES1 mRNA in the cells, which means that it suppressed Notch activity. The treatment up-regulated mRNA of nuclear factor kappa-B1 (NFKB1) and v-rel reticuloendotheliosis viral oncogene homolog A (RELA), which can be a cause of growth promotion. CONCLUSION: The diverse effects of GSIs must be elucidated before clinical use because they can stimulate the growth of leukemia cells.

Cyclopamine and quercetin suppress the growth of leukemia and lymphoma cells.

BACKGROUND: Hedgehog (Hh) and Wnt signaling pathways are involved in the stimulation of growth of leukemia and lymphoma cells. In the present study, whether or not the Hh inhibitor, cyclopamine, and the Wnt inhibitor, quercetin, suppress cell growth was investigated. MATERIALS AND METHODS: The effects of cyclopamine and quercetin on the in vitro growth and protein expression of ten acute leukemia and B-cell lymphoma cell lines were examined. RESULTS: Cyclopamine and quercetin suppressed cell growth and induced apoptosis in seven and eight cell lines respectively. Cyclopamine decreased the level of Gli1 protein, a target gene product of Hh signaling. Quercetin decreased the level of Notch1 protein and its active fragment in the DND-41 T-lymphoblastic leukemia cell line with constitutive Notch activation. CONCLUSION: Cyclopamine and quercetin suppress the growth of a number of leukemia and lymphoma cells. This finding suggests the potential use of these compounds in molecularly-targeted therapy for leukemia and lymphoma.