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Biomedical subjects

S C Prasad

Publications and source records attributed to S C Prasad.

At least 19 recordsLinked to original sources
PubMed1999

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 p/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.

Apoptosis↗
PubMed1999

Differential expression of stathmin during neoplastic conversion of human prostate epithelial cells is reversed by hypomethylating agent, 5-azacytidine.

In a variety of human tumor tissues, including those of prostate and breast, CpG hypermethylation represents one of the mechanisms downregulating the expression of specific proteins, including tumor suppressor proteins. Using 267B1-XR cells generated by ionizing radiation-induced transformation of epithelial cells, derived from neonatal human prostate and immortalized by SV40 (267B1), we now report markedly low levels of expression of the cytoplasmic phosphoprotein stathmin, in addition to several proteins of the actin microfilaments and intermediate filaments that characterize the altered phenotype. Stathmin is emerging as a relay protein integrating signals from diverse pathways during differentiation and neoplastic progression. In this in vitro prostate carcinogenesis model system, where loss of specific-protein expression is a major feature of the transformed 267B1-XR cells, we employed 5-azacytidine treatment followed by 2D-PAGE to reveal if experimental genomic hypomethylation reinstated the levels of any of the differentially expressed proteins. Our data suggest that stathmin represents one such example.

Antimetabolites, Antineoplastic↗
PubMed1998

Apoptosis-associated proteolysis of vimentin in human prostate epithelial tumor cells.

Vimentin intermediate filaments (IF) are responsible for regulation of cell attachment and subcellular organization. Using an in vitro model system of human prostate epithelial cells (267B1-XR), we demonstrate that a series of vimentin proteolytic fragments represent some of the differentially expressed proteins in 2D-gel profiles of the apoptotic cells undergoing ionizing radiation-induced cell death. A caspase-sensitive motif search suggests that the type III IF protein (vimentin) is subject to proteolysis to promote the execution phase of apoptosis, in a manner similar to the well-established type V (lamins) and type I (keratins 18, 19) IF proteins. Furthermore, vimentin and a few of its derived polypeptides, reported to be specific to the apoptotic process, correspond to ubiquinated proteins, thus pointing to the complex interrelationships of protein ubiquination in solubilizing the IF network during apoptosis.

Apoptosis↗
PubMed1998

Effects of collimator jaw setting on dose output for treatments with multileaf collimator.

The variations in dose output with collimator jaw setting have been investigated for treatment fields shaped by a multileaf collimator. Measurements have been made for 6 and 15 MV x-ray beams on a Varian Clinac 2100C machine. The results of our study show that the collimator jaw settings can affect the dose by about 5% for a small field shaped by a multileaf collimator. The effect is smaller for larger fields.

Humans↗
PubMed1998

Two-dimensional gel electrophoresis analyses identify nucleophosmin as an estrogen regulated protein associated with acquired estrogen-independence in human breast cancer cells.

We have used two-dimensional gel electrophoresis to identify proteins associated with estrogen-induced proliferation in MCF-7 breast cancer cells and their progression to estrogen-independent proliferation. We compared the total cellular proteins from MCF-7 cells and an estrogen independent derivative of the MCF-7 cells MCF-7/LCC1 (Brünner et al. Cancer Research 1993, 53, 283-290), each grown with and without estradiol. These comparisons reveal seven estrogen-regulated proteins. Three of these proteins (HI-1: 36 kDa/pI 4.5, HI-10: 40 kDa/pI 5.5 and HI-19: 62 kDa/pI 5.0) exhibit a 'progression-like' pattern, being induced by estradiol in MCF-7 cells and constitutively present/upregulated in the MCF-7/LCC1 growing without estradiol. HI-11 (65 kDa/pI 5.5) is strongly induced by estradiol in MCF-7 cells but constitutively downregulated and unresponsive to estradiol in the MCF-7/LCC1 cells. Two proteins exhibit a suppressor pattern and are downregulated by estradiol in the estrogen-dependent MCF-7 cells (HI-3: 44 kDa/pI 4.4 and HI-4: 56 kDa/ pI 5.2) and present in MCF-7/LCC1 cells growing without estradiol at levels comparable to that seen in estrogen-treated MCF-7 cells. One protein (HI-9: 68 kDa/pI 5.5) exhibits a marked estrogen regulated pI shift, rather than changes in abundance. We purified and sequenced the HI-10 protein, which we identified as the nucleolar protein, nucleophosmin (NPM). One- and two-dimensional Western blot analyses of MCF-7/LCC1 cell lysates confirmed that HI-10 is immunoreactive with an antinucleophosmin antibody. Western blotting also confirmed the estrogenic regulation of NPM seen in the initial two-dimensional gel electrophoresis studies. Thus, NPM is induced by estradiol in the MCF-7 cells and upregulated in the MCF-7/LCC1 cells growing without estrogen, clearly associating its expression with an acquired estrogen-independent phenotype. NPM has several potentially important roles in regulating cell function and signaling. It is a substrate for phosphorylation by p34cdc2 kinase, protein kinase C and nuclear kinase II, and a repressor of the transcriptional regulating activities of both the IRF-1 tumor suppressor protein and the YY1 transcription factor. Studies are currently underway to determine which of these NPM functions may be involved in the hormonal progression of breast cancer.

Amino Acid Sequence↗
PubMed1997

Laser-based microfocused x-ray source for mammography: feasibility study.

A laser-produced plasma (LPP) x-ray source with possible application in mammography was created by focusing a laser beam on a Mo target. A Table-Top-Terawatt (TTT) laser operating at 1 J energy per pulse was employed. A dual pulse technique was used. Maximum energy transfer (approximately 10%) from laser light to hot electrons was reached at a 150 ps delay between pulses and the conversion efficiency (hard x-ray yield/laser energy input) was approximately 2 x 10(-4). The created LPP x-ray source is characterized by a very small focal spot size (tens of microns), Gaussian brightness distribution, and a very short pulse duration (a few ps). The spectral distribution of the generated x rays was measured. Images of the focal spot, using a pinhole camera, and images of a resolution pattern and a mammographic phantom were obtained. The LPP focal spot modulation transfer function for different magnification factors was calculated. We have shown that the LPP source in conjunction with a spherically bent, high throughput, crystal monochromator in a fixed-exit Rowland circle configuration can be used to created a narrow band tunable mammography system. Tunability to a specific patient breast tissue thickness and density would allow one to significantly improve contrast and resolution (exceeding 20 lp/mm) while lowering the exposure up to 50% for thicker breasts. The prospects for the LPP x-ray source for mammographic application are discussed.

Biophysical Phenomena↗
PubMed1996

Radiation-induced neoplastic transformation of human prostate epithelial cells.

We report the malignant transformation of human prostate epithelial cells (267B1) after multiple exposures to ionizing radiation. Carcinogenic progression of cells from immortal growth to anchorage-independent growth in soft agar to tumorigenicity in athymic mice resulted after a cumulative X-ray dose of 30 Gy. The tumors were characterized histologically as poorly differentiated adenocarcinomas, expressed prostate-specific antigen, and stained positive for keratin. No p53 or ras mutations were observed. Numerous chromosomal defects were noted on karyotypes after radiation exposure. However, chromosome 3 and 8 translocations were observed predominantly in the tumor outgrowths. These findings provide the first evidence of malignant transformation of human prostate epithelial cells exposed to ionizing radiation.

Animals↗
PubMed1996

Comparison of measured and calculated dose for asymmetric x-ray beams defined by independently movable collimators.

Linear accelerators with x-ray collimators that move independently are becoming increasingly common for treatment with asymmetric fields. In an asymmetric field, the center of the treatment field is away from the true central axis where dosimetric data are normally obtained. In this paper we present a simplified approach to the calculation of dose for asymmetric fields. We use central axis tissue-maximum ratio, off-axis factor in phantom and relative field-size factor in phantom to calculate dose. The accuracy of our calculations has been compared with ion-chamber measurements for 6 and 15 MV x-ray beams. Measurements were made at 5, 10, and 15 cm off-axis for a 20 cm x 20 cm asymmetric field at dmax and 6 cm depths in a solid-water phantom using a 0.6 cc Farmer chamber. Agreement within 3% was found at the measurement points.

Equipment Design↗
PubMed1996

Scatter reduction in mammography with air gap.

Scatter reduction by air gaps in mammography was investigated. We have experimentally demonstrated that, independently of the imaging geometry, scatter in air-gap mammography can be well described by a virtual source of scatter (VSS) model. This model postulates that scatter radiation originates from a virtual point source of scatter placed on the central axis between the x-ray source and the exit surface of a patient at distance delta and utilizes only two parameters: delta and (S/P)0. The (S/P)0 parameter represents scatter-to-primary ratio without an air gap and delta is the distance from the exit surface of a patient to the virtual source of scatter. We have experimentally determined the analytical form of the two independent parameters of the VSS model; delta exhibits a linear increase proportional to the radiation field size, does not depend on patient thickness, and is in the 10-30 cm range, while (S/P)0 increases with the field size as a power function and is in the 0.4-1.3 range. In the framework of the VSS model the selectivity, the contrast improvement factor, and the signal-to-noise improvement factor were employed to evaluate performance of air-gap mammography systems. We have demonstrated that selectivity of an air gap rapidly deteriorates at some well-defined critical value of scatter fraction that has profound consequences on air-gap performance. Assuming fixed patient exposure, the results shows that, if a contrast limited detection system (such as film/screen mammography) is used, an air gap system can outperform a grid system only if a very large source-to-patient (SPD) distance is utilized, which might be possible with new laser-based x-ray sources. For the noise limited detection systems (such as digital mammography) even a small SPD (70 cm) and a small air-gap (20 cm) system will outperform a grid system.

Air↗
PubMed1996

Cytoskeletal changes during radiation-induced neoplastic transformation of human prostate epithelial cells.

We recently reported tumorigenic transformation of SV40-immortalized neonatal human prostate epithelial cells (267B1) by exposure to fractionated doses of X-rays. Altered morphology and anchorage independence were observed following two successive fractions of 2 Gy each (F3-SAC). Additional 2 Gy treatments to these non-tumorigenic cells to a total dose of 30 Gy resulted in radiation-transformed tumorigenic colonies (267B1-SXR). Malignant transformation of parental 267B1 cells was also achieved by consecutive 2 Gy exposures to a total dose of 30 Gy (267B1-XR). This study discusses the cytoskeletal changes in the F3-SAC, 267B1-XR and 267B1-SXR derivatives of these human prostate epithelial cells. Confocal and conventional fluorescence microscopy of filamentous actin showed numerous, well organized, evenly distributed stress fibers in the parental cells prior to irradiation, while the anchorage-independent cells and several tumorigenic derivatives exhibited poor stress fiber organization after radiation exposure. This disorganization of actin microfilaments in the radiation-transformed cells was also accompanied by changes in the expression of selective tropomyosin isoforms as judged by two-dimensional gel electrophoresis. These changes in actin organization and tropomyosin expression appear to be coincidental with morphological transformation and acquisition of tumorigenicity in the 267B1 cells following radiation exposure.

Actin Cytoskeleton↗
PubMed1995

Surface dose for tangentially incident photon beams in the presence of air gap and air cavity.

Dose to a surface parallel to an incident photon beam has been investigated in the presence of air gaps and air cavities. Measurements have been made in an acrylic phantom with a parallel plate chamber. Photon beams of Co 60, 4-MV, 6-MV, and 15-MV X-rays have been investigated. The results of our measurements show that in the case of air gaps, the dose reduction to a tangential surface is considerable. On the other hand, the dose reduction is 2% or less in the case of air cavity for volumes encountered in clinical situations.

Air↗
PubMed1993

A simple approach to the technical aspects of radiosurgery treatments.

An approach to radiosurgery treatment that can be readily adopted in most radiotherapy centers with linear accelerators is presented. In our institution, a Leksell-type of neurosurgical frame, a computed tomography scanner, locally fabricated cones, and 6 MV X-ray beams are used to perform radiosurgery treatments. Collimated arcs with dose distributions, that conform to the shape of the lesion in the transverse and the sagittal planes are used. It is argued that the uncertainties in the localization of the isocenter within a lesion and the specifications of the size of the target volume do not justify high precision mechanical devices for most radiosurgery treatments.

Brain Mapping↗
PubMed1993

Analysis of mannoproteins from blastoconidia and hyphae of Candida albicans with a common epitope recognized by anti-complement receptor type 2 antibodies.

Mannoproteins of approximately 50 kDa from blastoconidia and 60 kDa from hyphae of Candida albicans reacted in Western blots (immunoblots) with either a polyclonal rabbit antiserum (CA-7) or a monoclonal antibody (CA-A) to the C. albicans C3d-binding protein (complement receptor type 2). The glycosylated nature of these proteins was demonstrated by their reactivity with concanavalin A and by selective labeling with the biotin-hydrazide reagent following periodate oxidation. Differences in the oligosaccharides of these proteins were observed in regard to their reactivity with lectin-peroxidase reagents and sensitivity to glycosidases such as N-glycanase or endoglycosidase F (but not endoglycosidase H). The 60-kDa mannoprotein reacted with wheat germ agglutinin, while the 50-kDa mannoprotein did not. Treatment of the 60-kDa mannoprotein with the glycosidases mentioned above resulted in its conversion into a species of 40 to 45 kDa. Enzyme treatment had no obvious effect on the electrophoretic mobility of the 50-kDa species from blastoconidia. Both the 50- and 60-kDa glycoproteins remained immunoreactive after treatment with the glycosidases. Reactivities of the two mannoproteins to neuraminidase also differed. Finally, the 50-kDa (blastoconidia) and the 60-kDa (hyphae) mannoproteins were purified by using ion-exchange chromatography and electroelution. The purified proteins differed in net charge, the 60-kDa species having a more acidic pI. Functional activity of the purified mannoproteins was demonstrated, as each inhibited the rosetting of antibody-sensitized sheep erythrocytes conjugated with iC3b or C3d by hyphae. Thus, an epitope(s) common to both a mycelial and blastoconidial mannoprotein is associated with a structurally different oligosaccharide for each growth form.

Animals↗
PubMed1992

High-resolution two-dimensional electrophoresis of nuclear proteins: a comparison of HeLa nuclei prepared by three different methods.

A comparative analysis of HeLa cell nuclear proteins is presented using Iso-Dalt methods of protein resolution in two dimensions. The nuclear proteins were prepared by (1) spin through glycerol cushion, (2) spin through sucrose cushion, or (3) Triton wash. Improved resolution of total nuclear proteins in the range of pH 4.5-6.0 was achieved by substituting longer isotubes in combination with broad-range ampholines during the isoelectric focusing step. An attempt to indicate silver stainable protein spots common to total cellular extracts and nuclear preparations has been made. Also, proteins that appear to be well represented in all three nuclear preparations and remain undetectable in the total cellular protein pattern have been marked as probably being enriched nuclear proteins. Such a comparative analysis of whole nuclear protein preparations made it possible to document that the different preparations preserved the same set of proteins. The Triton-wash method of obtaining nuclei was identified as the preferred choice. Coomassie-stained gels and blots of these nuclear proteins could serve as a guide for accessing relevant protein spots for further biochemical analysis such as immunoblotting.

Antibody Specificity↗
PubMed1992

Comparison of absorbed doses in water and solid water for electron beams.

Solid water, as a substitute for water, has become commercially available for dosimetry measurements. A study was undertaken to compare the dose in water and solid water respectively for 6, 9, 12, 16 and 20 MeV electron beams. Measurements using ion chamber show that the dose in water is higher than the dose in solid water by 1% for 6, 9, and 12 MeV electrons. For 16 and 20 MeV electrons, the dose in water and solid water are the same within the uncertainty of our measurements.

Absorption↗
PubMed1991

Pitfalls in dose calculation using a commercial treatment planning computer for Clinac-4 4MV x-ray beam.

Dose calculations on a commercial treatment planning computer based on the storage of profile data along a principal axis for Clinac-4 4MV x-ray can lead to significant error in calculated dose in the corners of a large field. These errors are due to the unique nature of the lead flattening filter on Cl-4. A method is suggested to remedy this problem by storing profile data along a diagonal of a large field.

Humans↗
PubMed1990

Enhanced poly(adenosine diphosphate ribose) polymerase activity and gene expression in Ewing's sarcoma cells.

Ewing's sarcoma (ES) is a highly malignant childhood bone tumor and is considered curable by moderate doses of radiotherapy. The addition of chemical inhibitors of the activity of the nuclear enzyme poly(adenosine diphosphate ribose) [poly(ADPR)] polymerase to ES cells in culture results in increased cell killing, a phenomenon called "inhibitor sensitization." Since poly(ADPR) polymerase is thought to be associated with DNA repair, it has been suggested that ES cells and other inhibitor-sensitized cells may have a reduced capacity for polymer synthesis resulting in deficient postirradiation recovery. We present here the unexpected observation that in comparison to other cell lines tested, ES cells exhibit a high enzyme activity, higher constitutive levels of the protein, and elevated levels of its mRNA transcript for poly(ADPR) polymerase. No gross amplifications or rearrangements of the gene were observed; however, regulation of poly(ADPR) polymerase in these tumor cells takes place at the level of the gene transcript.

Blotting, Northern↗