Sarada C. Prasad

Protein changes associated with ionizing radiation‐induced apoptosis in human prostate epithelial tumor cells

Ionizing radiation (IR) is an important component in the therapy of localized prostate cancer. Identification of protein alterations during IR‐induced apoptosis prostate cancer cells is an important step toward understanding the new metabolic status of the dying cell. In the present study, we report changes in protein profile that define the execution phase of the apoptotic response in the in vitro model of tumorigenic radiation‐transformed SV40‐immortalized human prostate epithelial cells (267B1‐XR), induced to undergo programmed cell death by IR. We employed an approach that involves use of analytical two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) coupled with Western blotting with specific antisera. Our results point out that apoptotic cells experience significant reduction in the levels of the intermediate filament proteins, keratins‐18, 19, vimentin and the associated 14‐3‐3 adapter proteins. At the same time, molecular chaperones such as glucose‐regulated protein 94, calreticulin, calnexin, and protein disulfide isomerase exhibit marked accumulation in these dying cells. The present data indicate that apoptosis‐associated processes in prostate epithelial cells include solubilization of the rigid intermediate filament network by specific proteolysis as well as increased levels of endoplasmic reticulum (ER) proteins with chaperone functions.

Sodium butyrate induces apoptosis and accumulation of ubiquitinated proteins in human breast carcinoma cells

To investigate the possible relationship between apoptosis and the ubiquitin pathway we examined the patterns of ubiquitinated proteins in the human breast carcinoma MCF-7 cell line following induction of apoptotic death by sodium butyrate. Apoptosis in these cells was associated with internucleosomal DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase. By dual in situ antiubiquitin immunofluorescence and chromatin DNA staining, we demonstrated that ubiquitin fluorescence was increased specifically in cells that underwent sodium butyrate-mediated apoptosis. The extent of ubiquitin incorporation into protein conjugates was examined in both adherent (not yet apoptotic) and floating (apoptotic) cell populations. We found that apoptotic cells exhibited enhanced intensity of ubiquitin-immunoreactive conjugates, whereas adherent cells did not. In addition, two-dimensional immunoblot analysis of proteins from apoptotic cells identified a set of isomeric ubiquitinated conjugates located at a pI range of 4.2–4.6 and a Mr approximately of 30 kDa. These data indicate that the ubiquitin pathway may play a role in the sodium butyrate-induced apoptotic program in breast carcinoma cells.

Expression of cytokeratin-19 as a marker of neoplastic progression of human prostate epithelial cells

Our earlier studies demonstrated neoplastic transformation of SV40‐immortalized neonatal human prostate epithelial cells (267B1) by fractionated doses of ionizing radiation or by introduction of v‐ki‐ras oncogene. X‐ray‐treated 267B1 cells represent three different stages of neoplastic progression: nontumorigenic F3‐SAC cells that acquired morphological changes and anchorage independence when treated with 2 × 2 Gy of X‐rays; malignantly transformed 267B1‐XR and 267B1‐SXR cells that received 2‐Gy doses to a total of 30 Gy. We also reported alterations in cell size, morphology, actin stress fibers, and levels of actin‐binding proteins in these transformed human prostate cells.

A dot-blot method for screening polyclonal and monoclonal antisera to poly(ADP-ribose)

ADP-ribosylation reactions play a key role at several points in cellular regulation and repair of DNA damage. The use of polyclonal or monoclonal antisera to poly(ADP-ribose) as probes to localize the site(s) of action of the polymer offers a promising tool for these studies. We report here a simple, sensitive method for detection and titration of these antisera to poly(ADP-ribose) using nitrocellulose membrane (NC) as a support for a dot-blot analysis. We take advantage of the fact that a highly labeled poly(ADP-ribose) preparation can be obtained by incubation of a 0.3 M KCl extract prepared from calf thymus nuclei with 32P-NAD. Such a preparation of labeled antigen is used as a reagent to detect the positive antibody spots on the NC with negligible background. Subsequent titration of the antisera and their semi-quantitative evaluation are also feasible using the dot-blot method. The sensitivity of the assay is only limited by the specific activity that can be achieved for the labeled polymer prepared as the antigen probe. The advantage of this method is that it eliminates the need to prepare pure, highly radiolabeled polymer as well as the fact that several samples can be handled on the membrane simultaneously. We demonstrate application of this technique for screening sera from patients with systemic lupus erythematosus (SLE) for anti-poly(ADP-ribose) antibodies. Further, we also extend the use of these sera for immunoquantitation of ADP-ribosylated proteins in six human tumor cells in tissue culture.

Detection of heterogeneity of apoptotic fragments of poly (ADP‐ribose) polymerase in MDA‐MB‐468 breast cancer cells: Two‐dimensional gel analysis

Caspace‐mediated proteolysis of the nuclear enzyme poly(ADP‐ribose) polymerase (PARP) (EC 2.4, 2.30) is a biochemical marker of cell death in response to various apoptotic stimuli. Anti‐PARP antibodies identifying the 89 kDa polypeptide from the C‐terminus as well as the 113 kDa native enzyme are often used to demonstrate evidence of apoptosis‐associated, interleukin converting enzyme (ICE)‐mediated limited cleavage. Recent evidence points to redundancy of caspases, heterogeneity of their cleavage sites, and a possibility of generating distinct context‐specific, and cell‐specific PARP fragments. In the present study, we employed antibodies directed to multiple sites in PARP and probed two‐dimensionally resolved proteins of the estrogen receptor negative MDA‐MB‐468 breast tumor cells, induced to undergo apoptosis by ionizing radiation (IR). Our results revealed that the 24 kDa apoptotic fragment of PARP, from the N‐terminus, consists of at least three isoforms, located at a pI more basic than the full length enzyme. We also report a hitherto unrecognized feature of an anti‐PARP antiserum, VIC‐5, detecting both the 89 kDa and the 24 kDa caspase‐generated fragments of PARP. Thus, application of two‐dimensional electrophoresis combined with antisera directed to multiple sites would be valuable in distinguishing PARP cleavage site‐ and inhibitor specificities of proteases during apoptosis.

Cytoskeletal and adhesion protein changes during neoplastic progression of human prostate epithelial cells

In vitro model systems have facilitated the identification of altered cellular functions in transformed cells, including motility, attachment to the basement membrane and response to inter/intra-cellular signals. Recent studies on prostate cancer models and other transformed epithelial cell systems, have shown downregulation of several actin-binding proteins, such as gelsolin, tropomyosins, a-actinin, vinculin and myosin light chain-2 (MLC-2), along with an accompanying lack of well-organized actin stress fibers. The transformed phenotype in prostate epithelial cells is also characterized by alterations in the expression and function of the E-cadherin mediated cell–cell adhesion system. Most of these changes appear to depend upon the actin-based dynamic remodeling of prostate tumor cell acquiring special growth and invasive advantages. Furthermore, when the effects of reverse-transforming agents have been studied in vitro, the target proteins identified to date have been of cytoskeletal origin. This review examines contemporary prostate cancer research pointing to the tumor suppressive role of certain actinbinding and cell adhesion proteins and their correlations with enhanced cell motility, invasion and metastatic properties in prostate carcinoma in vitro and in vivo. * Corresponding author: Tel.: +1 202 6874901; fax: +1 202 6872221; e-mail: prasads@gunet.georgetown.edu

Quantitation of Strand Breaks in Human DNA Using 32P-Alu Hybridization: Application to Exponential and Plateau-Phase Cells

A sensitive quantitation of DNA (0.2 to 10 ng) can be achieved using a 32P-labeled Alu probe to hybridize human DNA spotted onto nylon membrane. This allows the determination of radiation-induced single-strand breaks without the use of [3H]thymidine prelabeling of cells in culture. The sensitivity of this technique in HeLa cells is comparable to results obtained using the alkaline unwinding technique. The method is applicable to cells in both exponential and plateau phases of growth.

1044 Molecular characterization of radiation-induced transformation of human prostate epithelial cells

It is generally accepted that the carcinogenic process in mammalian cell systems involves multiple steps that result in distinct phenotypic alterations associated with immortalization, progression, and conversion to malignancy. Prostate cancer, as the most common cancer in men, presents a particular challenge: not all histologic cancer progresses to clinical malignancies. The steps necessary for prostate carcinogenesis need to be defined and model systems expanded. We report the neoplastic transformation of an immortalized human prostate epithelial cell line (267Bl) by ionizing radiation.

Heterogeneity of Polyclonal Antisera to ADP-Ribose and Their Use as Probes for ADP-Ribosylation in Human Tumor Cells

DNA strand breaks, induced by exposure to chemicals or ionizing radiation stimulate poly(ADP-ribose) polymerase activity (1, 2). The resultant protein modifications have been postulated to comprise an important step in the DNA repair process (3). Inhibitors of the polymerase have been shown to sensitize human fibroblasts (4) and certain tumor cells (5) to ionizing radiation and to inhibit the repair of potentially lethal radiation injury (6, 7). That the response of the tumor cell lines vary, with some showing sensitivity to inhibitors of poly(ADP-ribosyl)ation and irradiation while others do not, suggested a need for detailed investigation of the ADP-ribosylation process in these tumor cell lines. In the present study we report the quantitative variations in protein-bound mono(ADP-ribose) levels as well as poly(ADP-ribose) polymerase activities and cellular NAD levels of various tumor cells. To this end, we also describe the development and characterization of polyclonal antisera to mono(ADP-ribose) and its potential use as a probe for studies of ADP-ribosylation.