Anti-apoptotic Bcl-2 Family Genes Research Articles

Tumor suppressor p53 mediates interleukin-6 expression to enable cancer cell evasion of genotoxic stress

The tumor suppressor p53 primarily functions as a mediator of DNA damage-induced cell death, thereby contributing to the efficacy of genotoxic anticancer therapeutics. Here, we show, on the contrary, that cancer cells can employ genotoxic stress-induced p53 to acquire treatment resistance through the production of the pleiotropic cytokine interleukin (IL)-6. Mechanistically, DNA damage, either repairable or irreparable, activates p53 and stimulates Caspase-2-mediated cleavage of its negative regulator mouse double minute 2 (MDM2) creating a positive feedback loop that leads to elevated p53 protein accumulation. p53 transcriptionally controls the major adenosine triphosphate (ATP) release channel pannexin 1 (Panx1), which directs IL-6 induction via a mechanism dependent on the extracellular ATP-activated purinergic P2 receptors as well as their downstream intracellular calcium (iCa2+)/PI3K/Akt/NF-ĸB signaling pathway. Thus, p53 silencing impairs Panx1 and IL-6 expression and renders cancer cells sensitive to genotoxic stress. Moreover, we confirm that IL-6 hampers the effectiveness of genotoxic anticancer agents by mitigating DNA damage, driving the expression of anti-apoptotic Bcl-2 family genes, and maintaining the migratory and invasive properties of cancer cells. Analysis of patient survival and relevant factors in lung cancer and pan-cancer cohorts supports the prognostic and clinical values of Panx1 and IL-6. Notably, IL-6 secreted by cancer cells during genotoxic treatments promotes the polarization of monocytic THP-1-derived macrophages into an alternative (M2-like) phenotype that exhibits impaired anti-survival activities but enhanced pro-metastatic effects on cancer cells as compared to nonpolarized macrophages. Our study reveals the precise mechanism for genotoxic-induced IL-6 and suggests that targeting p53-mediated IL-6 may improve the responsiveness of cancer cells to genotoxic anticancer therapy.

Venetoclax-Resistant T-ALL Cells Display Distinct Cancer Stem Cell Signatures and Enrichment of Cytokine Signaling

Therapy resistance remains one of the major challenges for cancer treatment that largely limits treatment benefits and patient survival. The underlying mechanisms that lead to therapy resistance are highly complicated because of the specificity to the cancer subtype and therapy. The expression of the anti-apoptotic protein BCL2 has been shown to be deregulated in T-cell acute lymphoblastic leukemia (T-ALL), where different T-ALL cells display a differential response to the BCL2-specific inhibitor venetoclax. In this study, we observed that the expression of anti-apoptotic BCL2 family genes, such as BCL2, BCL2L1, and MCL1, is highly varied in T-ALL patients, and inhibitors targeting proteins coded by these genes display differential responses in T-ALL cell lines. Three T-ALL cell lines (ALL-SIL, MOLT-16, and LOUCY) were highly sensitive to BCL2 inhibition within a panel of cell lines tested. These cell lines displayed differential BCL2 and BCL2L1 expression. Prolonged exposure to venetoclax led to the development of resistance to it in all three sensitive cell lines. To understand how cells developed venetoclax resistance, we monitored the expression of BCL2, BCL2L1, and MCL1 over the treatment period and compared gene expression between resistant cells and parental sensitive cells. We observed a different trend of regulation in terms of BCL2 family gene expression and global gene expression profile including genes reported to be expressed in cancer stem cells. Gene set enrichment analysis (GSEA) showed enrichment of cytokine signaling in all three cell lines which was supported by the phospho-kinase array where STAT5 phosphorylation was found to be elevated in resistant cells. Collectively, our data suggest that venetoclax resistance can be mediated through the enrichment of distinct gene signatures and cytokine signaling pathways.

The Role of Non-Coding RNAs in the Pathogenesis of AL Amyloidosis

Background: Systemic light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by deposition of misfolded immunoglobulin light chain products in vital organs, causing their dysfunction. All therapies used to treat AL patients are adapted from multiple myeloma (MM) and customized to the typically frail AL population. Hence, it is important to identify novel therapeutic targets for these patients. MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression and have a role in cancer development and progression. MiRNAs can be purified from serum, plasma and bone marrow (BM) and may be used as biomarkers to distinguish between patients and healthy individuals. Moreover, miRNA-mRNA interactions may determine the molecular mechanism involved in AL pathogenesis and may suggest novel therapeutic approaches. To date, knowledge about circulating or BM miRNAs involved in AL amyloidosis is lacking.Aims: To decipher specific miRNA expression profiles in AL amyloidosis compared to MM patients and healthy controls (HC) and to examine how miRNAs are involved in AL pathogenesis.Methods: miRNA expression profile was determined using the nCounter assay (NanoString technologies). RNA-Seq data was downloaded from GEO database (GSE175384), and was used to detect potential miRNA-mRNA targets, and enriched biological pathways by the bioinformatics tool Ingunity Pathway Analysis (IPA). MiRNA and gene expression profiles were validated by qRT-PCR in 60 AL, 60 MM and 10 HC samples. The effect of aberrantly expressed miRNAs on potential molecular targets was analyzed in ALMC1 cells by transfecting the cells with miRNA mimic, following qRT-PCR, Western blot analysis and Annexin-PI staining.Results: BM and plasma miRNAs were differentially expressed in AL amyloidosis compared to MM or HC. MiRs-9a-5p, 181a-5p, 199a-3p, 130a-3p, 145-5p and 301a-3p were differentially expressed between AL and MM samples and may be used as biomarkers for distinguish AL amyloidosis from MM. Moreover, we found that the differentially expressed miRNAs and mRNA in AL patients regulates key signaling pathways related to cell cycle and anti-apoptosis mechanisms including cytokine signaling, oxidative phosphorylation (OXPHOS), NFkB signaling, activation of MAPK and PI3K/AKT pathways, which are all linked to cancer cell growth, proliferation and therapeutic resistance, and therefore may be used as a therapeutic targets. Specifically, our analysis showed that genes related to mitochondrial activity were upregulated in AL patients (Figure 1), particularly the anti-apoptotic BCL2 family genes (BCL2, MCL1, and BCL2L1). MCL1 and BCL2L1 are predicted targets of miR-181a-5p that was downregulated in BM and plasma samples of AL patients compared to MM patients, indicating a possible interaction between these molecules. MiR-9-5p, which was also found to be downregulated in AL BM and plasma samples compared to MM patients, is predicted to have an indirect effect on the BCL2 family members through CREB molecule (Figure 1).The biological significance of miR-9-5p and miR-181a-5p was evaluated, by transfecting ALMC1 cell line with miRNA mimics. Overexpression of these miRNAs led to downregulation of the BCL2 family anti-apoptotic genes and induced apoptosis by Annexin V staining. These findings might explain the biological mechanism by which AL patients respond to the BCL2 inhibitor, venetoclax, as recently reported (Sidiqi et al, BCJ, 2020).Conclusions: We provide insight into the molecular mechanisms mediated by miRNAs and the aberrant expression of oncogenic/tumor suppressor genes. The differential expression of miRNAs in AL amyloidosis may be used to understand disease pathogenesis and predict risk of progression to AL amyloidosis among patients with known plasma cell disorders. Additionally, signaling pathways involved in AL amyloidosis, mediated by miRNAs, may assist in tailoring more specific treatments. [Display omitted] DisclosuresDispenzieri: Oncopeptides: Consultancy; Sorrento Therapeutics: Consultancy; Pfizer: Research Funding; Alnylam: Research Funding; Takeda: Research Funding; Janssen: Consultancy, Research Funding.

Cage Transcriptome Analysis Reveals BCL2A1 Upregulation in FLT3-ITD/D835 Dual Mutated AML Cells Harboring Complex Co-Mutations

Cage Transcriptome Analysis Reveals BCL2A1 Upregulation in FLT3-ITD/D835 Dual Mutated AML Cells Harboring Complex Co-Mutations

Abstract 1248: Not BCL2 but BFL1 is overexpressed and have strong correlation with NF-kB pathway in diffuse large B-cell lymphoma

Abstract Venetoclax, anti-apoptotic BCL2-selective inhibitor, has demonstrated efficacy in B-cell multiple lymphoid malignancies. However, several studies have shown that the other anti-apoptotic BCL2 family genes can increase the risk of venetoclax resistance. Accordingly, compounds targeting other BCL2 family are actively being developed. So it is necessary to find which BCL2 family genes is overexpressed in certain tumors. We profiled BCL2 family genes expression with RNA-seq across different types of lymphoid malignancies (follicular lymphoma, burkitt lymphoma, diffuse large b-cell lymphoma, multiple myeloma, and acute leukocyte leukemia). Log2-transformed TMM-normalized RPKM was used for expression levels. As is known, BCL2 and MCL1 was highly expressed in follicular lymphoma and multiple myeloma, respectively. Interestingly, we discovered BFL1, one of anti-apoptotic BCL2 family genes, is overexpressed in both activated B-cell (ABC) and germinal center B-cell (GCB) type diffuse large B cell lymphoma (DLBCL). It has been well known that BFL1 is a pro-survival NF-kB target gene and indeed NF-kB pathway is highly activated in ABC-DLBCL. Also recent studies have shown that activation of this pathway can occur in GCB-DLBCL. Using gene set enrichment analysis, we confirmed that high expression of BFL1 is associated with negative regulation of intrinsic apoptosis and activation of NF-kB in B-cell. Also we discovered that NF-kB pathway activation in correlation with BFL-1 is especially dominant in DLBCL compared to other lymphoid malignancies. Among NF-kB signal related molecules (TNF receptors or Toll-like receptors), CD40 expression significantly correlated with BFL1 expression. In contrast, BCL2, another important apoptosis related gene, showed a negative association with the NF-kB pathway in DLBCL. We could validate our observation using microarray data. In conclusion, BFL1, which may controls intrinsic apoptosis and have strong correlation with NF-kB, is overexpressed in DLCBL. We suggest not venetoclax but BFL1 inhibitor may exert clinical benefit in DLBCL. Citation Format: Chansub Lee, Hyojin Song, Seulki Song, Daeyoon Kim, Jihyun Park, Sungyoung Lee, Hongseok Yun, Sheehyun Kim, Sung-Soo Yoon, Youngil Koh. Not BCL2 but BFL1 is overexpressed and have strong correlation with NF-kB pathway in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1248.

Abstract 281: Sensitivity to splicing modulation of BCL2 family genes reveals cancer therapeutic strategies for splicing modulators

Abstract Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment. Citation Format: Daniel Aird, Teng Teng, Chia-Ling Huang, Ermira Pazolli, Deepti Banka, Kahlin Cheung-Ong, Cheryl Eifert, Craig Furman, Jeremy Wu, Michael Seiler, Silvia Buonamici, Peter Fekkes, Craig Karr, James Palacino, Eunice Park, Peter Smith, Lihua Yu, Yoshiharu Mizui, Markus Warmuth, Agustin Chicas, Laura Corson, Ping Zhu. Sensitivity to splicing modulation of BCL2 family genes reveals cancer therapeutic strategies for splicing modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 281.

Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.

Abstract IA06: Oncogenic translation programs

Abstract New York, NY. I will provide an overview of the lab's contribution to deciphering the role of aberrant mRNA translation in cancer. We initially reported that aberrant eIF4E expression can drive lymphomagenesis in vivo. Next, we noticed that eIF4E's oncogenic activity strictly required phosphorylation at S209 by MNK1/2 kinases. The latter is the basis of ongoing efforts to develop and test MNK kinase inhibitors for cancer applications. More recently, we dissected the molecular basis of the anti-cancer action of eIF4A inhibitors like silvestrol. Using the ribosome footprinting technology developed by the Weissman lab we noticed that eIF4A inhibition acutely and preferentially suppressed a select group of ~200 mRNAs. These are marked by repeated 5' UTR sequences that are predicted to fold into energetically favorable G-quadruplex structures. Notably, many of these eIF4A–dependent transcripts encode oncogenic transcription factors (e.g. MYC and MYB), a number of cell cycle regulators (e.g. CDK6, CCND3), and anti-apoptotic BCL2 family genes. These findings also begin to shed light on the overarching question of how aberrant translation may lead to cancer. We speculate that it is not simply a global increase in protein production but instead the activation of oncogenic translation programs. We will discuss these findings also in light of recent work proposing an additional mechanism of eIF4A action that emphasizes binding to ubiquitous RNA sequence elements over the enzymatic activity of eIF4A. Citation Format: Hans-Guido Wendel. Oncogenic translation programs. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA06.

Angiopoietin-like protein 2 renders colorectal cancer cells resistant to chemotherapy by activating spleen tyrosine kinase-phosphoinositide 3-kinase-dependent anti-apoptotic signaling.

Angiopoietin-like protein 2 (ANGPTL2) plays an important role in inflammatory carcinogenesis and tumor metastasis by activating tumor angiogenesis and tumor cell chemotaxis and invasiveness. However, it is unclear whether ANGPTL2 expression has an effect on tumor cell survival. Here, we explored that possibility by determining whether ANGPTL2 expression altered survival of human colorectal cancer cell lines treated with antineoplastic drugs. To do so, we generated SW480 cells expressing ANGPTL2 (SW480/ANGPTL2) and control (SW480/Ctrl) cells. Apoptosis induced by antineoplastic drug treatment was significantly decreased in SW480/ANGPTL2 compared to control cells. Expression of anti-apoptotic BCL-2 family genes was upregulated in SW480/ANGPTL2 compared to SW480/Ctrl cells. To assess signaling downstream of ANGPTL2 underlying this effect, we carried out RNA sequencing analysis of SW480/ANGPTL2 and SW480/Ctrl cells. That analysis, combined with in vitro experiments, indicated that Syk-PI3K signaling induced expression of BCL-2 family genes in SW480/ANGPTL2 cells. Furthermore, ANGPTL2 increased its own expression in a feedback loop by activating the spleen tyrosine kinase–nuclear factor of activated T cells (Syk–NFAT) pathway. Finally, we observed a correlation between higher ANGPTL2 expression in primary unresectable tumors from colorectal cancer patients who underwent chemotherapy with a lower objective response rate. These findings suggest that attenuating ANGPTL2 signaling in tumor cells may block tumor cell resistance to antineoplastic therapies.

STAT3-RANTES Autocrine Signaling Is Essential for Tamoxifen Resistance in Human Breast Cancer Cells

The acquisition of tamoxifen resistance is a major therapeutic problem in breast cancer. We developed a tamoxifen-resistant MCF-7 (TRM-7) cell line to elucidate the molecular mechanisms and factors associated with acquisition of such resistance. We showed that phosphorylation of STAT3 at tyrosine 705 (Y705) and RANTES expression are increased in response to tamoxifen in human breast cancer cells. On the basis of these results, we hypothesize that upregulated STAT3 phosphorylation and RANTES may be correlated with the development of drug resistance. Here, we showed that STAT3 and RANTES contribute to the maintenance of drug resistance. STAT3 phosphorylation is constitutively retained via a RANTES autocrine loop, which in turn upregulates anti-apoptotic signals in TRM-7 cells. STAT3-RANTES autocrine signaling affected expression of anti-apoptotic BCL-2 family genes and prevented TRM-7 cells from undergoing programmed cell death by inhibiting PARP and caspase-9 cleavage. Subsequently, blockade of STAT3 and RANTES in TRM-7 cells resulted in reduction of anti-apoptotic signals, which was rescued by exogenous RANTES treatment; drug resistance was also restored. Taken together, our results suggested that STAT3-RANTES autocrine signaling is essential for maintenance of drug resistance and inhibition of programmed cell death. These mechanisms of STAT3-RANTES autocrine signaling suggest a novel strategy for management of patients with tamoxifen-resistant tumors.

Longterm influence of hypoxic-ischemic brain damage in 3-day-old rats on their brain MR imaging and memory and learning ability

Objective To investigate the activation of apoptotic genes of the brain with hypoxia- ischemia (HI) in newborn SD rats, and MRI changes and memory and learning ability in adulthood. Methods HI was induced by right carotid artery ligation followed by 2.5 h of hypoxia (6% O2) on 3-day-old SD rats (n=36). Control pups were sham-operated (n = 27). Right brain hemisphere was collected at 12 h and 7 d after HI and subjected to an apoptosis Oligo GEArrayR. MRI and Morris water maze test were performed on both groups at 42 and 44 days old, respectively. Results Comparing to 12 h after HI, up-regulated apoptotic genes included TNF, Caspase and pro-apoptotit genes of Bcl2 families, whereas the anti-apoptotic genes of Bcl2 family were down-regulated at 7 d after HI. The MRI assessment of the rats in HI group demonstrated that the area of the right cerebra l cortex was significantly smaller than the left side and control [periventricular layer: (23.5±3.6) mm2 vs (33.0±4.3) mm2, (34.5±3.9) mm2; hippocampus layer: (18.9±4.4) mm2 vs (29.1±5.0) mm2,(30.8±4.5) mm2, both P<0.01]. During the navigation trial, the HI rats demonstrated longer escape latency (4th day: (52.7±35.9) vs (17.8±8. 9) s, P<0.01). HI rats passed the platform less times than the control ones (T= 292.5, P<0.05) in space probe trial. Conclusions The activation of apoptotic genes induced by HI brain injury remains until 7 days later, involving intrinsic and extrinsic apoptotic pathway. The apoptosis of neural cells may lead to poor development of the cortex and impair the memory and learning ability in the adult rats after neonatal hypoxia- ischemia injury. Key words: Hypoxia-ischemia, brain; Infant, premature; Apoptosis; Learning; Memory; Magnetic resonance imaging

Apoptotic gene expression in neuropathic pain

AbstractPain initiated or caused by a primary lesion or dysfunction in the nervous system is defined as neuropathic pain. It results from direct injury to nerves in the peripheral or central nervous system and is associated with several clinical symptoms. Neuropathic pain treatment is extremely difficult, as it is a very complex disease, involving several molecular pathways. Excitatory or inhibitory pathways controlling neuropathic pain development show altered gene expression, caused by peripheral nerve injury.This study used several experimental pain models to demonstrate the occurrence of programmed cell death in the centers controlling pain induction and maintenance, such as spinal cord and pre-frontal cortex. We combined behavioural, molecular and morphological approaches to assess the involvement of bcl-2 gene family and caspases in neuropathic pain. Chronic constriction injury (CCI) and spared nerve injury (SNI) of rodent sciatic nerve induced the appearance of pain-like behaviours, such as hyperalgesia and allodynia. An early (2-3 days post-CCI) apoptosis appeared in the spinal cord neurons as the pro-apoptotic bax gene increased (320±19%). The incidence of apoptosis appeared to be limited to the first few days following nerve injury. Subsequently, increased expression of anti-apoptotic bcl-2 family genes may inhibit further neuron loss. SNI triggered apoptotic pathway and caspases activation in pre-frontal cortex 7, 14, and 21 days post-injury. Among the time-points analyzed, RT-PCR analysis showed increased expression of the bax/bcl-2 ratio (40±2%), bid (16±2%), caspase-1 (84±3%), caspase-8 (53±6%), caspase-9 (25±6%), caspase-12 (58±2%), TNF (32±2%) genes in the cortex by 7 days post-injury. Western blot analysis showed increased active Caspase-3 protein levels in the cortex at 3, 7, 14, and 21 post-surgery. This study shows that apoptotic genes could be an useful pharmacological target in neuropathic pain controlling.

Apoptotic gene expression in neuropathic pain

Pain initiated or caused by a primary lesion or dysfunction in the nervous system is defined as neuropathic pain. It results from direct injury to nerves in the peripheral or central nervous system and is associated with several clinical symptoms. Neuropathic pain treatment is extremely difficult, as it is a very complex disease, involving several molecular pathways. Excitatory or inhibitory pathways controlling neuropathic pain development show altered gene expression, caused by peripheral nerve injury.This study used several experimental pain models to demonstrate the occurrence of programmed cell death in the centers controlling pain induction and maintenance, such as spinal cord and pre-frontal cortex. We combined behavioural, molecular and morphological approaches to assess the involvement of bcl-2 gene family and caspases in neuropathic pain. Chronic constriction injury (CCI) and spared nerve injury (SNI) of rodent sciatic nerve induced the appearance of pain-like behaviours, such as hyperalgesia and allodynia. An early (2-3 days post-CCI) apoptosis appeared in the spinal cord neurons as the pro-apoptotic bax gene increased (320±19%). The incidence of apoptosis appeared to be limited to the first few days following nerve injury. Subsequently, increased expression of anti-apoptotic bcl-2 family genes may inhibit further neuron loss. SNI triggered apoptotic pathway and caspases activation in pre-frontal cortex 7, 14, and 21 days post-injury. Among the time-points analyzed, RT-PCR analysis showed increased expression of the bax/bcl-2 ratio (40±2%), bid (16±2%), caspase-1 (84±3%), caspase-8 (53±6%), caspase-9 (25±6%), caspase-12 (58±2%), TNF (32±2%) genes in the cortex by 7 days post-injury. Western blot analysis showed increased active Caspase-3 protein levels in the cortex at 3, 7, 14, and 21 post-surgery. This study shows that apoptotic genes could be an useful pharmacological target in neuropathic pain controlling.

Human FLT3/FLK2 Targets Hematopoietic Stem Cells and Granulocyte/Macrophage Progenitors.

FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing long-term hematopoietic stem cells (LT-HSCs), but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. In order to test whether Flt3 expression can delineate such a developmental pathway also in human hematopoiesis, we have analyzed the expression of human Flt3 (hFlt3) in prospectively-purified human stem and progenitors (PNAS 2002) by utilizing 7-color FACS and a highly efficient xenograft systems. We have found that Flt3 expression in early hematopoiesis is completely different between human and mice: hCD34+hCD38-hCD90+Lin-LT-HSCs capable of long-term reconstitution in xenogeneic hosts uniformly express hFlt3, and its expression is upregulated through hCD34+hCD38+hCD45RA-hCD123+Lin-common myeloid progenitors (CMPs) to hCD34+hCD38+hCD45RA+hCD123+Lin-granulocyte/macrophage progenitors (GMPs), but hCD34+hCD38+hCD45RA-hCD123-Lin- megakaryocyte/erythrocyte progenitors (MEPs) shut off its expression in human. Furthermore, we have also demonstrated that hFlt3 signaling can prevent stem and progenitors from apoptotic cell death in vitro without any effects on lineage fate decision. Next, we tried to find key molecules for Flt3-Flt3 ligand (FL)-mediating anti-apoptotic effect. First, we tested expression pattern of anti-apoptotic Bcl-2 family genes in HSCs, CMPs, GMPs, MEPs and common lymphoid progenitors (CLPs) in human hematopoiesis. Mcl-1, an indispensable survival factor for murine hematopoiesis (Science, 2005), was also expressed at the highest level in human HSCs, whereas Bcl-2 and Bcl-xL was highly expressed in GMPs and MEPs, respectively. Next, we examined whether FL stimulation can upregulate the expression of Bcl-2 family genes in human purified HSCs and progenitors. FL significantly upregulated the expression of Mcl-1, but not of Bcl-2 or Bcl-xL in HSCs as well as CMPs and GMPs. In conclusion, our data show that the distribution of Flt3 is quite different in mouse and human hematopoeisis. Human Flt3 targets LT-HSCs and myeloid progenitors except for MEPs. Flt3 signaling might support cell survival in early hematopoiesis including the HSC and the myeloid progenitor stages through upregulation of Mcl-1. This is a striking example that the expression pattern of key molecules could be significantly different between human and mouse.

Genomics of Ischemia

The quest to understand the neurobiology of ischemic brain injury has relied on the investigational techniques available over the years. Historically, morbid anatomy was the approach used through the midportion of the last century.1 The establishment of the Journal of Neurochemisty in 1956 marks the assent of tools to understanding brain injury beyond the whole organ level. Cellular and molecular approaches supervened especially after the discovery of apoptosis or programmed cell death by Kerr and colleagues in 1972,2 which lead to a focus on cellular and molecular cell death mechanisms. We now know that ischemic cell death is accompanied by transcriptional and translational events that may modulate ischemic injury. The induction of death-inducing gene products, such as the caspases, and the regulation of prosurvival gene products, such as antiapoptotic bcl-2 family genes, are now well known.3 More broad-based approaches to gene modulation in disease, such as …

A forskolin derivative, FSK88, induces apoptosis in human gastric cancer BGC823 cells through caspase activation involving regulation of Bcl-2 family gene expression, dissipation of mitochondrial membrane potential and cytochrome c release

FSK88, a forskolin derivative, was extracted and purified from cultured tropical plant roots, Coleus forskohlii. Our previous studies have demonstrated that FSK88 can inhibit HL-60 cell proliferation and induce the differentiation of HL-60 cells to monocyte macrophages. In this study, we showed that FSK88 can induce apoptotic death of human gastric cancer BGC823 cells in a dose- and time-dependent manner. Results showed that FSK88-induced apoptosis was accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in BGC823 cells. Furthermore, treatment with caspase-3 inhibitor (z-DEVD-fmk) was capable of preventing the FSK88-induced caspase-3 activity and apoptosis. FSK88-induced apoptosis in human gastric cancer BGC823 cells was also accompanied by the up-regulation of Bax, Bad and down-regulation of Bcl-2. Theses results clearly demonstrated that the induction of apoptosis by FSK88 involved multiple cellular and molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family genes, mitochondrial membrane potential (Deltapsi(m)), cytochrome c, and caspase-3, participate in the FSK88-induced apoptotic process in human gastric cancer BGC823 cells.