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Jialing Xiang

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Narcisa Martinez Quiles

Universidad Complutense de Madrid

Meenakshi A . Chellaiah

University of Maryland Baltimore

Majid Ghassemian

Monica Naujokas

McGill University

Klemens Rottner

JOSE MANUEL MARTIN VILLA

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Papers by Jialing Xiang

The C-terminus of Ubl4A is critical for pro-death activity and association with the Arp2/3 complex

Biochemical and biophysical research communications, Jan 23, 2018

Ubl4A is a small ubiquitin-like protein involved in diverse cellular functions. We have shown tha... more Ubl4A is a small ubiquitin-like protein involved in diverse cellular functions. We have shown that Ubl4A is critical for survival of the starvation-mediated cell death in vivo. The underlying mechanism for this is through interaction with the actin-related protein Arp2/3 complex and promotion of actin branching. Interestingly, "put-back" of Ubl4A to Ubl4A-deficient cells also results in cell death. Removal of the Ubl4A N-terminus significantly enhances its cytotoxicity, indicating that the pro-death activity of Ubl4A is mainly from its C-terminal region. In vitro protein pull-down assays show that the C-terminal region of Ubl4A can directly interact with the Arp2/3 complex. The single point mutation of an aspartic acid to alanine (D122A) in the Ubl4A C-terminus abolishes its ability to bind the Arp2/3 complex. This mutation also destabilizes Ubl4A proteins susceptible to protease degradation. Importantly, ectopic expression of wild-type Ubl4A can induce cell death in colon...

A Structural Model for Bax∆2-Mediated Activation of Caspase 8-Dependent Apoptosis

International Journal of Molecular Sciences

Bax∆2 is a pro-apoptotic anti-tumor protein in the Bax family. While most of the Bax family cause... more Bax∆2 is a pro-apoptotic anti-tumor protein in the Bax family. While most of the Bax family causes cell death by targeting mitochondria, Bax∆2 forms cytosolic aggregates and activates caspase 8-dependent cell death. We previously showed that the Bax∆2 helix α9 is critical for caspase 8 recruitment. However, the interaction between these two proteins at the structural level is unknown. In this in silico study, we performed molecular dynamics (MD) simulations and protein–protein docking on Bax∆2 variants. The results suggest that the Bax∆2 variants have different stable states. Mutating the Baxα mitochondria-targeting signal [L26P/L27P] appears to introduce a kink into helix α1. Protein–protein docking suggests that helices α9 of both wild-type Bax∆2 and Bax∆2 caspase 8 binding-deficient mutant [L164P] can fit in the same caspase 8 binding site, but the mutant is unable to fit as well as wild-type Bax∆2. Together, these data point to a structural basis for explaining Bax∆2 function in...

Expression profile of the proapoptotic protein Bax in the human brain

Histochemistry and Cell Biology

Quantification of cell surface receptor in live tissue culture using a paired-agent stain and rinse approach

Biomedical Optics 2016, 2016

A fluorescence imaging approach is presented to quantify cancer cell-surface receptor concentrati... more

Detection of melanin-mediated false-positive for BaxΔ2 immunohistochemical staining in human skin tissues

BaxΔ2 is a pro-apoptotic isoform of the Bax family. We have previously shown that BaxΔ2 protein e... more BaxΔ2 is a pro-apoptotic isoform of the Bax family. We have previously shown that BaxΔ2 protein expression is low in most healthy human tissues with the protein mainly scattered in the connective tissues. Surprisingly, in skin tissue, the BaxΔ2-positive staining was strikingly strong, especially in the basal layer of the epidermis. It has been documented that melanin in the basal cells displays a brown color that could give false-positive staining in the commonly used DAB-based (3-3’-diaminobenzidine) immunostaining. To avoid the false-positive staining from DAB-melanin, we performed immunofluorescent staining on the same set of tissues which were positive for BaxΔ2 in the DAB immunostaining. The results from co-immunofluorescent staining of BaxΔ2 and a basal cell marker CK-14 showed that the previously detected DAB-stained BaxΔ2-positive epidermal cells were CK-14 positive but BaxΔ2 negative. Consistent with our previous finding, the BaxΔ2-positive cells in the dermis connective ti...

Rinsing paired-agent model (RPAM) to quantify cell-surface receptor concentrations in topical staining applications of thick tissues

Physics in medicine and biology, Jan 21, 2017

Conventional molecular assessment of tissue through histology, if adapted to fresh thicker sample... more Conventional molecular assessment of tissue through histology, if adapted to fresh thicker samples, has the potential to enhance cancer detection in surgical margins and monitoring of 3D cell culture molecular environments. However, in thicker samples, substantial background staining is common despite repeated rinsing, which can significantly reduce image contrast. Recently, 'paired-agent' methods-which employ co-administration of a control (untargeted) imaging agent-have been applied to thick-sample staining applications to account for background staining. To date, these methods have included (1) a simple ratiometric method that is relatively insensitive to noise in the data but has accuracy that is dependent on the staining protocol and the characteristics of the sample; and (2) a complex paired-agent kinetic modeling method that is more accurate but is more noise-sensitive and requires a precise serial rinsing protocol. Here, a new simplified mathematical model-the rinsin...

Quantification of cell surface receptor expression in live tissue culture media using a dual-tracer stain and rinse approach

Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XIII, 2015

Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecule... more Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, “untargeted” imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.

JNK Suppresses Apoptosis via Phosphorylation of the Proapoptotic Bcl-2 Family Protein BAD

Molecular Cell, 2004

JNK has been suggested to be proapoptotic, antiapoptotic, or have no role in apoptosis depending ... more JNK has been suggested to be proapoptotic, antiapoptotic, or have no role in apoptosis depending on the cell type and stimulus used. The precise mechanism of JNK action, under conditions when it promotes cell survival, is not entirely clear. Here, we report that JNK is required for IL-3-mediated cell survival through phosphorylation and inactivation of the proapoptotic Bcl-2 family protein BAD. IL-3 withdrawal-induced apoptosis is promoted by inhibition of JNK but suppressed by expression of a constitutively active JNK. JNK phosphorylates BAD at threonine 201, thereby inhibiting BAD association with the antiapoptotic molecule BCL-X(L). IL-3 induces BAD phosphorylation at threonine 201, and replacement of threonine 201 by alanine generates a BAD mutant, which promotes IL-3 withdrawal-induced apoptosis. Thus, our results provide a molecular mechanism by which JNK contributes to cell survival.

Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage

Cell Research, 2007

Prostate cancer is a frequently diagnosed cancer in men. The growth of prostate cancer is initial... more Prostate cancer is a frequently diagnosed cancer in men. The growth of prostate cancer is initially androgendependent and androgen ablation is a leading therapeutic option for prostate cancer, especially metastatic cancer [1, 2]. However, malignant prostate cancer eventually relapses and grows independently of androgen. The molecular mechanism by which androgen-dependent prostate cancer cells acquire the ability to grow under the conditions of androgen-deprivation is poorly understood. Bcl-2 is an anti-apoptotic oncoprotein, whose oncogenic effects are mediated by preventing cell death, rather than

Cyclic AMP inhibits JNK activation by CREB-mediated induction of c-FLIPL and MKP-1, thereby antagonizing UV-induced apoptosis

Cell Death & Differentiation, 2008

The cyclic AMP (cAMP) signaling pathway has been reported to either promote or suppress apoptosis... more The cyclic AMP (cAMP) signaling pathway has been reported to either promote or suppress apoptosis, in a cell contextdependent manner. Our previous study has shown that cAMP, by protein kinase A (PKA)-cAMP response element-binding protein (CREB)-dynein light chain (DLC) pathway, negatively regulates mitogen-activated protein kinase p38 activation, thereby contributing to tumor necrosis factor (TNF)-a-induced apoptosis in certain types of cells. However, it remains largely unknown how cAMP suppresses apoptosis. Here we report that cAMP antagonized UV-induced apoptosis in Rat-1 and NIH 3T3 cells. Despite that cAMP significantly suppressed UV-induced p38 activation, inhibition of p38 activity showed no significant effect on UV-induced cell death in both cell lines. Further studies revealed that cAMP antagonized UV-induced apoptosis by inhibition of c-Jun N-terminal protein kinase (JNK) activation. The induction of the long form of cellular FLICE-inhibitory protein (c-FLIP L) and mitogen-activated protein kinase phosphatase-1 (MKP-1), but not DLC and p21 WAF1 by CREB was required for cAMP-mediated inhibition of JNK activation. The suppression by cAMP of UV-induced apoptosis was reversed by c-FLIP L small-interfering RNA (siRNA) or MKP-1 siRNA, which released the inhibition of JNK activation by cAMP. Thus, our results provide a molecular mechanism by which cAMP suppresses JNK activation and antagonizes apoptosis.

Inactivation of BAD by IKK Inhibits TNFα-Induced Apoptosis Independently of NF-κB Activation

Cell, 2013

The IkB kinase complex (IKK) is a key regulator of immune responses, inflammation, cell survival,... more The IkB kinase complex (IKK) is a key regulator of immune responses, inflammation, cell survival, and tumorigenesis. The prosurvival function of IKK centers on activation of the transcription factor NF-kB, whose target gene products inhibit caspases and prevent prolonged JNK activation. Here, we report that inactivation of the BH3-only protein BAD by IKK independently of NF-kB activation suppresses TNFa-induced apoptosis. TNFa-treated Ikkb À/À mouse embryonic fibroblasts (MEFs) undergo apoptosis significantly faster than MEFs deficient in both RelA and cRel due to lack of inhibition of BAD by IKK. IKK phosphorylates BAD at serine-26 (Ser26) and primes it for inactivation. Elimination of Ser26 phosphorylation promotes BAD proapoptotic activity, thereby accelerating TNFainduced apoptosis in cultured cells and increasing mortality in animals. Our results reveal that IKK inhibits TNFa-induced apoptosis through two distinct but cooperative mechanisms: activation of the survival factor NF-kB and inactivation of the proapoptotic BH3-only BAD protein.

Detection of pro-apoptotic Bax∆2 proteins in the human cerebellum

Histochemistry and Cell Biology, 2018

BaxΔ2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsa... more BaxΔ2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, BaxΔ2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of BaxΔ2 expression in human tissues, we examined a panel of human brain samples. Here, we report four cerebellar cases in which the subjects had no neurological disorder or disease documented. We found BaxΔ2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong BaxΔ2-positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of BaxΔ2 is consistent with that known for Baxα, but in a less ubiquitous manner, these results may imply a potential function of BaxΔ2 in Purkinje cells.

Molecular cloning and expression of alternatively spliced PITSLRE protein kinase isoforms

Journal of Biological Chemistry, 1994

* This work was supported by National Institutes of Health Grant GM 44088 (to V. J. K.), by Cance... more * This work was supported by National Institutes of Health Grant GM 44088 (to V. J. K.), by Cancer Center Core Grant CA 21765 (to St. Jude Children's Research Hospital), and by support from the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The PITSLRE protein kinase family

Progress in Cell Cycle Research, 1995

A family of p34Cdc2 related protein kinases, the PITSLRE kinases, is generated by alternative spl... more A family of p34Cdc2 related protein kinases, the PITSLRE kinases, is generated by alternative splicing and promoter utilization from three duplicated and tandemly linked genes on human chromosome 1p36.3, which is frequently deleted during the late stages of tumorigenesis. PITSLRE mRNA, protein, and enzyme activity are induced during Fas receptor- and glucocorticoid-mediated apoptosis of human T cells. Several PITSLRE isoforms are specific targets of proteolysis during apoptosis, generating an enzymatically active 50 kDa isoform. Inhibition of this protease activity blocks PITSLRE processing and enzyme activation, as well as apoptosis. Thus, PITSLRE kinases may be integral downstream components of apoptotic signal transduction pathway(s). Furthermore, PITSLRE genes, and their products, are physically altered in human neuroblastoma tumors, suggesting that they may be tumor suppressors.

Cell Cycle-Related Protein Kinases and T Cell Death

Advances in Experimental Medicine and Biology, 1995

Programmed cell death (PCD), or apoptosis, involves the activation of a specific suicide program ... more Programmed cell death (PCD), or apoptosis, involves the activation of a specific suicide program within a cell. PCD is responsible for such diverse activities as the elimination of cells during normal embryological development and determination of the immune receptor repertoire (1–9). Even though PCD was first experimentally defined over twenty years ago, very little is known about the molecular events involved in this process. Emerging data indicate that there are multiple ways by which apoptosis can be triggered in various cell types. It is not yet known whether these triggers share a common pathway, or multiple pathways that share one or more components. Several genes have been cloned from C. elegans that are clearly involved in PCD during development (ced genes), but many of their functions have been inferred by analysis of the predicted protein structure and by genetic disruptions (8). Recent work has revealed the function of at least two ced genes. The ced-3 gene is the C. elegans homologue of the mammalian interleukin-1β-converting enzyme (ICE), and encodes a cysteine protease (10). ced-9 encodes the C. elegans homologue of the bcl-2 gene product (11). From these studies it is apparent that certain aspects of PCD have been well conserved evolutionarily.

Alterations in the PITSLRE protein kinase gene complex on chromosome 1p36 in childhood neuroblastoma

Nature Genetics, 1994

Detection of Bax Microsatellite Mutations and BaxÃÂ2 Isoform in Human Buccal Cells

Journal of Cell Science & Therapy, 2015

Loss of the pro-apoptotic Bcl-2 family protein Bax occurs in ~50% of hereditary nonpolyposis colo... more Loss of the pro-apoptotic Bcl-2 family protein Bax occurs in ~50% of hereditary nonpolyposis colorectal cancer (HNPCC) due to microsatellite instability (MSI). Recently, we found that some of the "Bax-negative" MSI tumor cells contain a functional Bax isoform, BaxΔ2, which sensitizes cells to selective chemotherapeutics. Here we show the detection of Bax microsatellite mutations and expression of BaxΔ2 proteins in human buccal cells. Our study provides a sensitive and noninvasive approach and a potential clinical application in diagnosis and treatment of MSI colon cancer patients.

Androgen via p21 Inhibits Tumor Necrosis Factor α-induced JNK Activation and Apoptosis

Journal of Biological Chemistry, 2009

The male hormone androgen is a growth/survival factor for its target tissues or organs. Yet, the ... more The male hormone androgen is a growth/survival factor for its target tissues or organs. Yet, the underlying mechanism is incompletely understood. Here, we report that androgen via p21 inhibits tumor necrosis factor ␣-induced JNK activation and apoptosis. Inhibition by androgen requires the transcription activity of androgen receptor (AR) and de novo protein synthesis. Androgen⅐AR induces expression of p21 that in turn inhibits tumor necrosis factor ␣-induced JNK and apoptosis. Furthermore, genetic interruption of p21 alleles abolishes the inhibition by androgen. Our results reveal a novel cross-talk between androgen⅐AR and JNK, thereby providing a molecular mechanism underlying the survival function of androgen.

Ubl4A is critical for mitochondrial fusion process under nutrient deprivation stress

Mitochondrial fusion and fission are dynamic processes regulated by the cellular microenvironment... more Mitochondrial fusion and fission are dynamic processes regulated by the cellular microenvironment. Under nutrient starvation conditions, mitochondrial fusion is strengthened for energy conservation. We have previously shown that newborns of Ubl4A-deficient mice were more sensitive to starvation stress with a higher rate of mortality than their wild-type littermates. Ubl4A binds with the actin-related protein Arp2/3 complex to synergize the actin branching process. Here, we showed that deficiency in Ubl4A resulted in mitochondrial fragmentation and apoptosis. A defect in the fusion process was the main cause of the mitochondrial fragmentation and resulted from a shortage of primed Arp2/3 complex pool around the mitochondria in the Ubl4A-deficient cells compared to the wild-type cells. As a result, the mitochondrial fusion process was not undertaken quickly enough to sustain starvation stress-induced cell death. Consequently, fragmented mitochondria lost their membrane integrity and R...

Detection of Pro-apoptotic BaxΔ2 Proteins in the Human Cerebellum

BaxΔ2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsa... more BaxΔ2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, BaxΔ2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of BaxΔ2 expression in human tissues, we examined a panel of human brain samples. Here, we report 4 cerebellar cases in which the subjects had no neurological disorder or disease documented. We found BaxΔ2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong BaxΔ2- positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. B...

The C-terminus of Ubl4A is critical for pro-death activity and association with the Arp2/3 complex

Biochemical and biophysical research communications, Jan 23, 2018

Ubl4A is a small ubiquitin-like protein involved in diverse cellular functions. We have shown tha... more Ubl4A is a small ubiquitin-like protein involved in diverse cellular functions. We have shown that Ubl4A is critical for survival of the starvation-mediated cell death in vivo. The underlying mechanism for this is through interaction with the actin-related protein Arp2/3 complex and promotion of actin branching. Interestingly, "put-back" of Ubl4A to Ubl4A-deficient cells also results in cell death. Removal of the Ubl4A N-terminus significantly enhances its cytotoxicity, indicating that the pro-death activity of Ubl4A is mainly from its C-terminal region. In vitro protein pull-down assays show that the C-terminal region of Ubl4A can directly interact with the Arp2/3 complex. The single point mutation of an aspartic acid to alanine (D122A) in the Ubl4A C-terminus abolishes its ability to bind the Arp2/3 complex. This mutation also destabilizes Ubl4A proteins susceptible to protease degradation. Importantly, ectopic expression of wild-type Ubl4A can induce cell death in colon...

A Structural Model for Bax∆2-Mediated Activation of Caspase 8-Dependent Apoptosis

International Journal of Molecular Sciences

Bax∆2 is a pro-apoptotic anti-tumor protein in the Bax family. While most of the Bax family cause... more Bax∆2 is a pro-apoptotic anti-tumor protein in the Bax family. While most of the Bax family causes cell death by targeting mitochondria, Bax∆2 forms cytosolic aggregates and activates caspase 8-dependent cell death. We previously showed that the Bax∆2 helix α9 is critical for caspase 8 recruitment. However, the interaction between these two proteins at the structural level is unknown. In this in silico study, we performed molecular dynamics (MD) simulations and protein–protein docking on Bax∆2 variants. The results suggest that the Bax∆2 variants have different stable states. Mutating the Baxα mitochondria-targeting signal [L26P/L27P] appears to introduce a kink into helix α1. Protein–protein docking suggests that helices α9 of both wild-type Bax∆2 and Bax∆2 caspase 8 binding-deficient mutant [L164P] can fit in the same caspase 8 binding site, but the mutant is unable to fit as well as wild-type Bax∆2. Together, these data point to a structural basis for explaining Bax∆2 function in...

Expression profile of the proapoptotic protein Bax in the human brain

Histochemistry and Cell Biology

Quantification of cell surface receptor in live tissue culture using a paired-agent stain and rinse approach

Biomedical Optics 2016, 2016

A fluorescence imaging approach is presented to quantify cancer cell-surface receptor concentrati... more

Detection of melanin-mediated false-positive for BaxΔ2 immunohistochemical staining in human skin tissues

BaxΔ2 is a pro-apoptotic isoform of the Bax family. We have previously shown that BaxΔ2 protein e... more BaxΔ2 is a pro-apoptotic isoform of the Bax family. We have previously shown that BaxΔ2 protein expression is low in most healthy human tissues with the protein mainly scattered in the connective tissues. Surprisingly, in skin tissue, the BaxΔ2-positive staining was strikingly strong, especially in the basal layer of the epidermis. It has been documented that melanin in the basal cells displays a brown color that could give false-positive staining in the commonly used DAB-based (3-3’-diaminobenzidine) immunostaining. To avoid the false-positive staining from DAB-melanin, we performed immunofluorescent staining on the same set of tissues which were positive for BaxΔ2 in the DAB immunostaining. The results from co-immunofluorescent staining of BaxΔ2 and a basal cell marker CK-14 showed that the previously detected DAB-stained BaxΔ2-positive epidermal cells were CK-14 positive but BaxΔ2 negative. Consistent with our previous finding, the BaxΔ2-positive cells in the dermis connective ti...

Rinsing paired-agent model (RPAM) to quantify cell-surface receptor concentrations in topical staining applications of thick tissues

Physics in medicine and biology, Jan 21, 2017

Conventional molecular assessment of tissue through histology, if adapted to fresh thicker sample... more Conventional molecular assessment of tissue through histology, if adapted to fresh thicker samples, has the potential to enhance cancer detection in surgical margins and monitoring of 3D cell culture molecular environments. However, in thicker samples, substantial background staining is common despite repeated rinsing, which can significantly reduce image contrast. Recently, 'paired-agent' methods-which employ co-administration of a control (untargeted) imaging agent-have been applied to thick-sample staining applications to account for background staining. To date, these methods have included (1) a simple ratiometric method that is relatively insensitive to noise in the data but has accuracy that is dependent on the staining protocol and the characteristics of the sample; and (2) a complex paired-agent kinetic modeling method that is more accurate but is more noise-sensitive and requires a precise serial rinsing protocol. Here, a new simplified mathematical model-the rinsin...

Quantification of cell surface receptor expression in live tissue culture media using a dual-tracer stain and rinse approach

Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XIII, 2015

Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecule... more Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, “untargeted” imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.

JNK Suppresses Apoptosis via Phosphorylation of the Proapoptotic Bcl-2 Family Protein BAD

Molecular Cell, 2004

JNK has been suggested to be proapoptotic, antiapoptotic, or have no role in apoptosis depending ... more JNK has been suggested to be proapoptotic, antiapoptotic, or have no role in apoptosis depending on the cell type and stimulus used. The precise mechanism of JNK action, under conditions when it promotes cell survival, is not entirely clear. Here, we report that JNK is required for IL-3-mediated cell survival through phosphorylation and inactivation of the proapoptotic Bcl-2 family protein BAD. IL-3 withdrawal-induced apoptosis is promoted by inhibition of JNK but suppressed by expression of a constitutively active JNK. JNK phosphorylates BAD at threonine 201, thereby inhibiting BAD association with the antiapoptotic molecule BCL-X(L). IL-3 induces BAD phosphorylation at threonine 201, and replacement of threonine 201 by alanine generates a BAD mutant, which promotes IL-3 withdrawal-induced apoptosis. Thus, our results provide a molecular mechanism by which JNK contributes to cell survival.

Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage

Cell Research, 2007

Prostate cancer is a frequently diagnosed cancer in men. The growth of prostate cancer is initial... more Prostate cancer is a frequently diagnosed cancer in men. The growth of prostate cancer is initially androgendependent and androgen ablation is a leading therapeutic option for prostate cancer, especially metastatic cancer [1, 2]. However, malignant prostate cancer eventually relapses and grows independently of androgen. The molecular mechanism by which androgen-dependent prostate cancer cells acquire the ability to grow under the conditions of androgen-deprivation is poorly understood. Bcl-2 is an anti-apoptotic oncoprotein, whose oncogenic effects are mediated by preventing cell death, rather than

Cyclic AMP inhibits JNK activation by CREB-mediated induction of c-FLIPL and MKP-1, thereby antagonizing UV-induced apoptosis

Cell Death & Differentiation, 2008

The cyclic AMP (cAMP) signaling pathway has been reported to either promote or suppress apoptosis... more The cyclic AMP (cAMP) signaling pathway has been reported to either promote or suppress apoptosis, in a cell contextdependent manner. Our previous study has shown that cAMP, by protein kinase A (PKA)-cAMP response element-binding protein (CREB)-dynein light chain (DLC) pathway, negatively regulates mitogen-activated protein kinase p38 activation, thereby contributing to tumor necrosis factor (TNF)-a-induced apoptosis in certain types of cells. However, it remains largely unknown how cAMP suppresses apoptosis. Here we report that cAMP antagonized UV-induced apoptosis in Rat-1 and NIH 3T3 cells. Despite that cAMP significantly suppressed UV-induced p38 activation, inhibition of p38 activity showed no significant effect on UV-induced cell death in both cell lines. Further studies revealed that cAMP antagonized UV-induced apoptosis by inhibition of c-Jun N-terminal protein kinase (JNK) activation. The induction of the long form of cellular FLICE-inhibitory protein (c-FLIP L) and mitogen-activated protein kinase phosphatase-1 (MKP-1), but not DLC and p21 WAF1 by CREB was required for cAMP-mediated inhibition of JNK activation. The suppression by cAMP of UV-induced apoptosis was reversed by c-FLIP L small-interfering RNA (siRNA) or MKP-1 siRNA, which released the inhibition of JNK activation by cAMP. Thus, our results provide a molecular mechanism by which cAMP suppresses JNK activation and antagonizes apoptosis.

Inactivation of BAD by IKK Inhibits TNFα-Induced Apoptosis Independently of NF-κB Activation

Cell, 2013

The IkB kinase complex (IKK) is a key regulator of immune responses, inflammation, cell survival,... more The IkB kinase complex (IKK) is a key regulator of immune responses, inflammation, cell survival, and tumorigenesis. The prosurvival function of IKK centers on activation of the transcription factor NF-kB, whose target gene products inhibit caspases and prevent prolonged JNK activation. Here, we report that inactivation of the BH3-only protein BAD by IKK independently of NF-kB activation suppresses TNFa-induced apoptosis. TNFa-treated Ikkb À/À mouse embryonic fibroblasts (MEFs) undergo apoptosis significantly faster than MEFs deficient in both RelA and cRel due to lack of inhibition of BAD by IKK. IKK phosphorylates BAD at serine-26 (Ser26) and primes it for inactivation. Elimination of Ser26 phosphorylation promotes BAD proapoptotic activity, thereby accelerating TNFainduced apoptosis in cultured cells and increasing mortality in animals. Our results reveal that IKK inhibits TNFa-induced apoptosis through two distinct but cooperative mechanisms: activation of the survival factor NF-kB and inactivation of the proapoptotic BH3-only BAD protein.

Detection of pro-apoptotic Bax∆2 proteins in the human cerebellum

Histochemistry and Cell Biology, 2018

BaxΔ2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsa... more BaxΔ2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, BaxΔ2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of BaxΔ2 expression in human tissues, we examined a panel of human brain samples. Here, we report four cerebellar cases in which the subjects had no neurological disorder or disease documented. We found BaxΔ2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong BaxΔ2-positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of BaxΔ2 is consistent with that known for Baxα, but in a less ubiquitous manner, these results may imply a potential function of BaxΔ2 in Purkinje cells.

Molecular cloning and expression of alternatively spliced PITSLRE protein kinase isoforms

Journal of Biological Chemistry, 1994

* This work was supported by National Institutes of Health Grant GM 44088 (to V. J. K.), by Cance... more * This work was supported by National Institutes of Health Grant GM 44088 (to V. J. K.), by Cancer Center Core Grant CA 21765 (to St. Jude Children's Research Hospital), and by support from the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The PITSLRE protein kinase family

Progress in Cell Cycle Research, 1995

A family of p34Cdc2 related protein kinases, the PITSLRE kinases, is generated by alternative spl... more A family of p34Cdc2 related protein kinases, the PITSLRE kinases, is generated by alternative splicing and promoter utilization from three duplicated and tandemly linked genes on human chromosome 1p36.3, which is frequently deleted during the late stages of tumorigenesis. PITSLRE mRNA, protein, and enzyme activity are induced during Fas receptor- and glucocorticoid-mediated apoptosis of human T cells. Several PITSLRE isoforms are specific targets of proteolysis during apoptosis, generating an enzymatically active 50 kDa isoform. Inhibition of this protease activity blocks PITSLRE processing and enzyme activation, as well as apoptosis. Thus, PITSLRE kinases may be integral downstream components of apoptotic signal transduction pathway(s). Furthermore, PITSLRE genes, and their products, are physically altered in human neuroblastoma tumors, suggesting that they may be tumor suppressors.

Cell Cycle-Related Protein Kinases and T Cell Death

Advances in Experimental Medicine and Biology, 1995

Programmed cell death (PCD), or apoptosis, involves the activation of a specific suicide program ... more Programmed cell death (PCD), or apoptosis, involves the activation of a specific suicide program within a cell. PCD is responsible for such diverse activities as the elimination of cells during normal embryological development and determination of the immune receptor repertoire (1–9). Even though PCD was first experimentally defined over twenty years ago, very little is known about the molecular events involved in this process. Emerging data indicate that there are multiple ways by which apoptosis can be triggered in various cell types. It is not yet known whether these triggers share a common pathway, or multiple pathways that share one or more components. Several genes have been cloned from C. elegans that are clearly involved in PCD during development (ced genes), but many of their functions have been inferred by analysis of the predicted protein structure and by genetic disruptions (8). Recent work has revealed the function of at least two ced genes. The ced-3 gene is the C. elegans homologue of the mammalian interleukin-1β-converting enzyme (ICE), and encodes a cysteine protease (10). ced-9 encodes the C. elegans homologue of the bcl-2 gene product (11). From these studies it is apparent that certain aspects of PCD have been well conserved evolutionarily.

Alterations in the PITSLRE protein kinase gene complex on chromosome 1p36 in childhood neuroblastoma

Nature Genetics, 1994

Detection of Bax Microsatellite Mutations and BaxÃÂ2 Isoform in Human Buccal Cells

Journal of Cell Science & Therapy, 2015

Loss of the pro-apoptotic Bcl-2 family protein Bax occurs in ~50% of hereditary nonpolyposis colo... more Loss of the pro-apoptotic Bcl-2 family protein Bax occurs in ~50% of hereditary nonpolyposis colorectal cancer (HNPCC) due to microsatellite instability (MSI). Recently, we found that some of the "Bax-negative" MSI tumor cells contain a functional Bax isoform, BaxΔ2, which sensitizes cells to selective chemotherapeutics. Here we show the detection of Bax microsatellite mutations and expression of BaxΔ2 proteins in human buccal cells. Our study provides a sensitive and noninvasive approach and a potential clinical application in diagnosis and treatment of MSI colon cancer patients.

Androgen via p21 Inhibits Tumor Necrosis Factor α-induced JNK Activation and Apoptosis

Journal of Biological Chemistry, 2009

The male hormone androgen is a growth/survival factor for its target tissues or organs. Yet, the ... more The male hormone androgen is a growth/survival factor for its target tissues or organs. Yet, the underlying mechanism is incompletely understood. Here, we report that androgen via p21 inhibits tumor necrosis factor ␣-induced JNK activation and apoptosis. Inhibition by androgen requires the transcription activity of androgen receptor (AR) and de novo protein synthesis. Androgen⅐AR induces expression of p21 that in turn inhibits tumor necrosis factor ␣-induced JNK and apoptosis. Furthermore, genetic interruption of p21 alleles abolishes the inhibition by androgen. Our results reveal a novel cross-talk between androgen⅐AR and JNK, thereby providing a molecular mechanism underlying the survival function of androgen.

Ubl4A is critical for mitochondrial fusion process under nutrient deprivation stress

Mitochondrial fusion and fission are dynamic processes regulated by the cellular microenvironment... more Mitochondrial fusion and fission are dynamic processes regulated by the cellular microenvironment. Under nutrient starvation conditions, mitochondrial fusion is strengthened for energy conservation. We have previously shown that newborns of Ubl4A-deficient mice were more sensitive to starvation stress with a higher rate of mortality than their wild-type littermates. Ubl4A binds with the actin-related protein Arp2/3 complex to synergize the actin branching process. Here, we showed that deficiency in Ubl4A resulted in mitochondrial fragmentation and apoptosis. A defect in the fusion process was the main cause of the mitochondrial fragmentation and resulted from a shortage of primed Arp2/3 complex pool around the mitochondria in the Ubl4A-deficient cells compared to the wild-type cells. As a result, the mitochondrial fusion process was not undertaken quickly enough to sustain starvation stress-induced cell death. Consequently, fragmented mitochondria lost their membrane integrity and R...

Detection of Pro-apoptotic BaxΔ2 Proteins in the Human Cerebellum

BaxΔ2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsa... more BaxΔ2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, BaxΔ2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of BaxΔ2 expression in human tissues, we examined a panel of human brain samples. Here, we report 4 cerebellar cases in which the subjects had no neurological disorder or disease documented. We found BaxΔ2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong BaxΔ2- positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. B...