Byung Rho Lee

Improved Immunodetection of Endogenous α-Synuclein

α-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative α-synucleinopathies such as Parkinsons disease. Despite extensive research, however, its precise function remains unclear partly because of a difficulty in immunoblotting detection of endogenous α-synuclein. This difficulty has largely restricted the progress for α-synucleinopathy research. Here, we report that α-synuclein monomers tend to easily detach from blotted membranes, resulting in no or very poor detection. To prevent this detachment, a mild fixation of blotted membranes with paraformaldehyde was applied to the immunoblotting method. Amazingly, this fixation led to clear and strong detection of endogenous α-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous α-synuclein in various human cell lines, including SH-SY5Y, HEK293, HL60, HeLa, K562, A375, and Daoy, and a mouse cell line B16 as well as in several mouse tissues such as the spleen and kidney. Moreover, it should be noted that we could clearly detect endogenous α-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous α-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous α-synuclein, thereby facilitating α-synuclein research.

Role of Ser129 phosphorylation of α-synuclein in melanoma cells

Summary &agr;-Synuclein, a protein central to Parkinsons disease, is frequently expressed in melanoma tissues, but not in non-melanocytic cutaneous carcinoma and normal skin. Thus, &agr;-synuclein is not only related to Parkinsons disease, but also to melanoma. Recently, epidemiologists reported co-occurrence of melanoma and Parkinsons disease in patients, suggesting that these diseases could share common pathogenetic components and that &agr;-synuclein might be one of these. In Parkinsons disease, phosphorylation of &agr;-synuclein at Ser129 plays an important role in the pathobiology. However, its role in melanoma is not known. Here, we show the biological relevance of Ser129 phosphorylation in human melanoma cells. First, we have identified an antibody that reacts with Ser129-unphosphorylated &agr;-synuclein but not with Ser129-phosphorylated &agr;-synuclein. Using this and other antibodies to &agr;-synuclein, we investigated the role of Ser129 phosphorylation in human melanoma SK-MEL28 and SK-MEL5 cells. Our immunofluorescence microscopy showed that the Ser129-phosphorylated form, but not the Ser129-unphosphorylated form, of &agr;-synuclein localizes to dot-like structures at the cell surface and the extracellular space. Furthermore, immuno-electron microscopy showed that the melanoma cells release microvesicles in which Ser129-phosphorylated &agr;-synuclein localizes to the vesicular membrane. Taken together, our studies suggest that the phosphorylation of Ser129 leads to the cell surface translocation of &agr;-synuclein along the microtubule network and its subsequent vesicular release in melanoma cells.

IkB‐α‐specific transcript regulation by the C‐terminal end of c‐Rel

The NF‐kB family transcription factor c‐Rel is a critical molecule for inducing expression of cytokine genes by T cells. Here, we report that a deletion of the C‐terminal end, similar to the deletion in the highly oncogenic chicken v‐Rel gene, renders c‐Rel hyperactive toward cytokine gene promoters. At the same time, this mutation dramatically reduced c‐Rel activity in induction of IkB‐α mRNA expression. Moreover, ectopic expression of IkB‐α, along with the C‐terminal truncated c‐Rel, abrogates hyperactivity of this mutant. IkB‐α co‐expression did not affect the function of wild‐type c‐Rel. The data demonstrate that the C‐terminal end of c‐Rel has specific activity for IkB‐α mRNA expression and is dispensable for IL‐2 gene expression.

TIMP-1 Inhibits Apoptosis in Lung Adenocarcinoma Cells via Interaction with Bcl-2

Tissue inhibitors of metalloproteinases (TIMPs) are multifaceted molecules that exhibit properties beyond their classical proteinase inhibitory function. Although TIMP-1 is a known inhibitor of apoptosis in mammalian cells, the mechanisms by which it exerts its effects are not well-established. Our earlier studies using H2009 lung adenocarcinoma cells, implanted in the CNS, showed that TIMP-1 overexpressing H2009 cells (HB-1), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in the context of apoptosis, using the same lung cancer cell lines. Overexpressing TIMP-1 in a lung adenocarcinoma cell line H2009 resulted in an approximately 3-fold increased expression of Bcl-2, with a marked reduction in apoptosis upon staurosporine treatment. This was an MMP-independent function as a clone expressing TIMP-1 mutant T2G, lacking MMP inhibition activity, inhibited apoptosis as strongly as TIMP1 overexpressing clones, as determined by inhibition of PARP cleavage. Immunoprecipitation of Bcl-2 from cell lysates also co-immunoprecipitated TIMP-1, indicative of an interaction between these two proteins. This interaction was specific for TIMP-1 as TIMP-2 was not present in the Bcl-2 pull-down. Additionally, we show a co-dependency of TIMP-1 and Bcl-2 RNA and protein levels, such that abrogating Bcl-2 causes a downregulation of TIMP-1 but not TIMP-2. Finally, we demonstrate that TIMP-1 dependent inhibition of apoptosis occurs through p90RSK, with phosphorylation of the pro-apoptotic protein BAD at serine 112, ultimately reducing Bax levels and increasing mitochondrial permeability. Together, these studies define TIMP-1 as an important cancer biomarker and demonstrate the potential TIMP-1 as a crucial therapeutic target.

A simple and sensitive method for the demonstration of norepinephrine-storing adrenomedullary chromaffin cells

The medulla of the adrenal gland is a neuroendocrine tissue in which catecholamine-storing chromaffin cells exist. The chromaffin cells are derived from neural crest cells and distinctly differentiated into two types of cells, epinephrine (E) (adrenaline)-storing and norepinephrine (NE) (noradrenaline)-storing cells. Using histochemical or immunostaining methods, the two types of chromaffin cells have been differentially distinguished. However, difficulties and/or drawbacks of the procedures have somewhat restricted the progress of research in differential functions of E-storing and NE-storing cells. Here, we show a new method for the differential demonstration of these two cell types. We found that mouse and rat adrenomedullary cells are heterogeneously stained with Harris hematoxylin after treatment with citrate buffer at pH 6. The cell clusters stained with hematoxylin were positive for tyrosine hydroxylase, which is an enzyme involved in catecholamine biosynthesis. Furthermore, the cell clusters were negative for phenylethanolamine-N-methyl transferase, which is an enzyme responsible for the conversion from NE to E and expresses in E-storing chromaffin cells. Moreover, we found that the cell clusters stained with hematoxylin can also be stained with nitroblue tetrazolium at pH 11, using Hopsu and Mäkinen’s method by which NE-storing chromaffin cells are stained. These observations indicate that the cytoplasm of NE-storing chromaffin cells is specifically stained with hematoxylin after treatment with citrate buffer at pH 6. This method will allow us to facilitate cell-type specific research of chromaffin cells. Indeed, this method revealed that α-synuclein selectively expresses in E-storing chromaffin cells, but not in NE-storing chromaffin cells.

TIMP-1 downregulation modulates miR-125a-5p expression and triggers the apoptotic pathway

Matrix metalloproteinases and their natural inhibitors (TIMPs) are important elements in a wide range of oncology settings. Elevated levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) have often been associated with increased tumorigenesis. This has been demonstrated in a number of clinical and experimental models which include breast, gastric, colorectal and non-small cell lung carcinoma (NSCLC). Our earlier studies have identified increased angiogenic activity and aggressive tumor kinetics in TIMP-1 overexpressing H2009 lung adenocarcinoma cells. TIMP-1 overexpression has also been implicated in antiapoptotic responses, inducing a significant upregulation of Bcl-2. These TIMP-1 functions have been shown to be MMP-independent and provide insight into its pleiotropic activities. The current study examines microRNA (miRNA) interactions with this molecule. We have sought to define the relationship between TIMP-1 and miRNA by knocking down TIMP-1 in high TIMP-1 expressing lung adenocarcinoma cell lines. TIMP-1 knockdown resulted in increased expression of miR-125a-5p with a concomitant increase in apoptosis and attenuation of the tumorigenic features of these cells. We have identified TIMP-1 as a bona fide target of miR-125a-5p, and their interaction resulted in an increase in p53 expression. We further corroborated our in vitro data with patient samples, which exhibited an inverse correlation between TIMP-1 and miR-125a-5p expression. Our study lends support to the notion that elevated TIMP-1 levels, which are frequently associated with poor prognosis, cause aberrant modulation of miRNAs.

Abstract 3368: TIMP-1 and angiogenesis: A role for CDH5 and eNOS

Tissue inhibitors of metalloproteinases (TIMP) have emerged as diverse molecules with novel roles in apoptosis, angiogenesis and metastasis. TIMP-1, a well-documented inhibitor of apoptosis, has been shown to inhibit or promote angiogenesis. In recent years, a strong association has also been demonstrated between high levels of TIMP-1 and poor prognosis in many cancers. It has been recognized as a multifunctional protein affecting cell growth and angiogenesis. Conflicting studies have shown it to function either as a negative or a positive regulator of angiogenesis. In earlier studies, we have documented that lung adenocarcinoma cell line H2009, when transfected to overexpress TIMP-1 and injected into nude mice resulted in larger, more aggressive tumors with increased microvessel density, which was supported by in vitro angiogenesis assays whereby enhanced capillary network formation was seen. We have also shown that TIMP-1 expression levels can be correlated with KRAS independency in non-small-cell lung carcinoma (NSCLC) cell lines harboring KRAS mutations. The present study was undertaken to address the role of TIMP-1 in angiogenesis in the context of KRAS in NSCLC cell lines. KRAS dependent cell line H2009 and its TIMP-1 overexpressing clone HB1 and a KRAS independent cell line A549 and its TIMP-1 knockdown (KD) clone SH3 were examined by angiogenesis specific PCR array. Comparison of the angiogenesis associated profiles of these cell lines identified a marked increase in vascular endothelial cadherin CDH5 in TIMP-1 over-expressing clone HB1. KRAS independent A549 cells expressed high level of CDH5, however its TIMP-1 KD clone exhibited a marked decrease in CDH5 level. A similar profile was identified for endothelial nitric oxide synthase (eNOS/NOS3). CDH5 is an indispensable element of normal vascular development and maintenance, and any perturbations of normal levels will impact vessel sprouting and growth. eNOS is primarily active through production of nitric oxide, maintaining vascular tone as well as expressing anti-proliferative and antithrombotic properties. We confirmed our earlier observation that serum free conditioned media (SFCM) from TIMP-1 overexpressing HBI cells caused increased and more complex network formation. SFCM from A549 TIMP-1 KD SH3 clone showed a marked inhibition of endothelial network formation compared to SFCM from A549 cells. To confirm that TIMP-1 was responsible for the changes observed we purified secretory TIMP-1 by combined fast protein liquid chromatography and gel filtration from another high TIMP-1 producing NSCLC cell line (H460) and also observed more capillary network formation in human umbilical vein endothelial cells. Our studies suggest that TIMP-1 modulates angiogenesis by impacting CDH5 and eNOS positively. These results define an important function of TIMP-1 in angiogenesis and provide additional therapeutic targets for managing NSCLC. Citation Format: Sampa Ghoshal-Gupta, Ian A. Coe, Ammar Kutiyanawalla, Byung R. Lee, Amyn M. Rojiani, Mumtaz V. Rojiani. TIMP-1 and angiogenesis: A role for CDH5 and eNOS. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3368.

Abstract A49: Biological impact of TIMP-1 relative to KRAS status in lung carcinoma

Comprehensive sequence analysis of common human cancers has identified several genes that promote tumorigenesis. KRAS is a commonly mutated oncogene in lung adenocarcinoma, most frequently harboring mutations in codon 12 or 13. Therapeutically targeting of KRAS with small molecules has proven ineffective due to difficulties in disrupting protein/protein interactions that are inherent in its activation and function. Recent work has established that many cancers may have KRAS addiction and are considered KRAS dependent while others do not require KRAS for survival. Previously we have shown that TIMP-1 plays an important role in cell survival in lung adenocarcinoma cells. We have overexpressed TIMP-1 in H2009, a KRAS dependent cell line, which rendered these cells resistant to Staurosporine induced apoptosis, whereas knocking down TIMP-1 in A549 (a KRAS independent lung carcinoma cell line) had the opposite effect. We also noted that TIMP-1 overexpression in H2009 cells made them more angiogenic and aggressive in in vitro assays. H2009 and A549 cell lines also showed significant differences in migration, spheroid formation as well as invasiveness. Although H441 cell line is known to be KRAS dependent, it was not as invasive as H2009. These cell lines also showed differential expression of Vimentin and E-cadherin. These results prompted us to review available data regarding these cell lines, which not only harbored different mutations in the cancer promoting genes, but also differed at the regulatory level and functioned through activated ERK1/2, NFkB and Akt pathways. Analysis with the R2 program (http://hgserver1.amc.nl/cgi-bin/r2/) identified a significant correlation between TIMP-1 and KRAS in breast cancers and lung cancer . Interestingly, high TIMP1 levels have been shown to cause chemoresistance by virtue of its anti-apoptotic activity and KRAS has generally been undruggable. Our current study compares KRAS dependent and independent cell lines in relation to TIMP-1 expression. Modulating TIMP-1 renders these cells either more sensitive or resistant to apoptosis. Clinically, patients with elevated TIMP-1 expression have been shown to have shorter overall survival. This study helps to further dissect these pathways and provides a better understanding of the impact of these mutations in lung carcinoma. Supported in part by a Georgia Research Alliance Distinguished Cancer Scientist Award. Citation Format: Sampa Ghoshal Gupta, Ammar Kutiyanawalla, Byung Rho Lee, Amyn M. Rojiani, Mumtaz V. Rojiani. Biological impact of TIMP-1 relative to KRAS status in lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A49. doi: 10.1158/1557-3125.RASONC14-A49

Abstract 5269: Impact of K-ras status on differential expression of TIMP-1 in lung adenocarcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tissue inhibitor of metalloproteinase-1 exhibits a range of novel functions in addition to its initially recognized activity as a physiological inhibitor of MMPs. Two specific activities have been of significant interest in the context of oncology - inhibition of apoptosis and induction of cell proliferation. Previously we have shown that over-expressing TIMP-1 in a lung adenocarcinoma cell line H2009 results in more aggressive and larger tumors in vivo with increased invasion and colony formation in vitro. Subsequently we demonstrated elevated levels of Bcl-2 with a concomitant reduction in apoptosis induced by staurosporine. In the present study, we have sought to define a relationship between TIMP-1 expression levels in non-small-cell-lung-carcinoma (NSCLC) cell lines in relation to K-ras mutation. We identified a differential expression pattern of TIMP-1 in various NSCLC cell lines and also found that TIMP-1 overexpressing cell lines also had higher levels of Bcl-2 and vice versa. Upon further analysis, we were able to correlate a low TIMP-1 expression with K-ras dependence and high levels of E-cadherin expression as in H2009 and H441. Similarly, K-ras independent cell line A549, expressed low levels of E-cadherin and elevated TIMP-1. E-cadherin distribution was confirmed in A549 and H2009 cells by immunofluorescence. Since N-cadherin cleavage has been shown to be inhibited by TIMP-1, we examined N-cadherin levels by western blot. Results indicate that A549 exhibiting high levels of TIMP-1 had increased levels of N-cadherin whereas the low TIMP-1 expressing cell lines had lower N-cadherin levels. A spheroid formation assay to determine the aggressiveness of cell lines revealed that K-ras dependent cell line formed spheroids that were smaller and less migratory than K-ras independent cell line whereas the K-ras independent cell line A549 formed spheroids that were larger, more spread out exhibiting wider migratory properties. The present study underscores the multiple biologic properties of TIMP-1. It provides further mechanistic understanding of its documented role as a prognostic marker and a potential therapeutic target in various malignancies, particularly lung carcinoma. This study was supported in part by a Distinguished Cancer Scientist Award to AMR from the Georgia Research Alliance. Citation Format: Ammar Kutiyanawalla, Sampa Gupta-Ghoshal, Byung Rho Lee, Ravindra Kolhe, Amyn M. Rojiani, Mumtaz V. Rojiani. Impact of K-ras status on differential expression of TIMP-1 in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5269. doi:10.1158/1538-7445.AM2014-5269