PLD Protein-Protein Interactions With Signaling Molecules and Modulation by PA.

We describe methods for studying phospholipase D (PLD) interactions with signaling proteins and modulation of these interactions by the PLD reaction product, phosphatidic acid (PA). PLD is fundamental to the physiological maintenance of cellular/intracellular membranes, protein trafficking, cytoskeletal dynamics, membrane remodeling, cell proliferation, meiotic division and sporulation. PA is an acidic phospholipid involved in the biosynthesis of many other lipids that affects the enzymatic activities of many different signaling proteins via protein-lipid interactions or as a substrate. The involvement of PLD as an effector of protein-protein interactions and downstream signaling via PA-mediated processes has led to the investigation of PA-binding domains in target protein partners. We present here data and protocols detailing the interaction between PLD2-Rac2 interaction and modulation of this interaction by PA. We describe biochemical techniques to measure interactions between PLD, PA, and the small GTPase Rac2, which are associated in the cell. We found two maxima concentrations of PA that contributed to association or dissociation of Rac2 with PLD2, as well as the PLD2 lipase and guanine nucleotide exchange factor (GEF) activities. Fluctuations in the Rac2-PLD2 protein-protein binding interaction facilitate shuttling of Rac2 and/or PLD2 within the cell dependent on local cellular PA concentration. Fluorescence resonance emission transfer stoichiometry for PLD2 and Rac2 binding yielded a 3:1 ratio of Rac2:PLD2. Detection of PA in mammalian cells with a new biosensor showed colocalization in and around the nucleus. We also described methods for quantitation of PA in biological materials by HPLC electrospray ionization tandem mass spectrometry.

Phospholipase D (PLD) drives cell invasion, tumor growth and metastasis in a human breast cancer xenograph model.

Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based short hairpin RNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, P<0.05) and their onset delayed when compared with control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (5-fluoro-2-indolyl des-chlorohalopemide and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide). These inhibitors led to significant (>70%, P<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid, Wiscott-Aldrich Syndrome protein, growth receptor-bound protein 2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows for the first time that PLD2 has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target.

Cisplatin-DNA adducts inhibit translocation of the Ku subunits of DNA-PK.

We have determined the effect of cisplatin-DNA damage on the ability of the DNA-dependent protein kinase (DNA-PK) to interact with duplex DNA molecules in vitro. The Ku DNA binding subunits of DNA-PK display a reduced ability to translocate on duplex DNA containing cisplatin-DNA adducts compared to control, undamaged duplex DNA. The decreased rates of translocation resulted in a decrease in the association of the p460 catalytic subunit of DNA-PK (DNA-PKcs) with the Ku-DNA complex. In addition to a decrease in DNA-PKcs association, the DNA-PKcs that is bound with Ku at a DNA end containing cisplatin-DNA adducts has a reduced catalytic rate compared to heterotrimeric DNA-PK assembled on undamaged DNA. The position of the cisplatin-DNA lesion from the terminus also effects kinase activation, with maximal inhibition occurring when the lesion is closer to the terminus. These results are consistent with a model for DNA-PK activation where the Ku dimer translocates away from the DNA terminus and facilitates the association of DNA-PKcs which interacts with both Ku and DNA resulting in kinase activation. The presence of cisplatin adducts decreases the ability to translocate away from the terminus and results in the formation of inactive kinase complexes at the DNA terminus. The results are discussed with respect to the ability of cisplatin to sensitize cells to DNA damage induced by ionizing radiation and the ability to repair DNA double-strand breaks.

Cisplatin-induced apoptosis proceeds by caspase-3-dependent and -independent pathways in cisplatin-resistant and -sensitive human ovarian cancer cell lines.

We have assessed in detail the effect of cisplatin-activated programmed cell death in the cisplatin-sensitive human ovarian cancer cell line A2780 and two drug-resistant subclones, CP70 and C30. To determine whether the differential extent of apoptosis observed between the sensitive and resistant ovarian cancer cell lines was the result of dissimilar upstream signaling events, we assessed the execution of apoptotic events that precede target protein proteolysis and subsequent chromosomal DNA degradation. Proteolytic degradation of procaspase-3 was observed in both the CP70 and C30 cells following IC50 cisplatin treatment, whereas no proteolyzed caspase-3 subunits were detected in the A2780 cells. However, using a direct enzymatic assay measuring cleavage of the synthetic peptide substrate (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide), activity was detected in extracts prepared from A2780 cells treated at the IC90 level of cisplatin and was 2-3-fold less than that of extracts prepared from CP70 and C30 cells. Because the activation of procaspase-3 by caspase-9 requires the release of cytochrome c into the cytoplasm, we determined the level of cytoplasmic cytochrome c in each cell line in response to cisplatin treatment. Consistent with the caspase-3 activation data, a very small increase in cytoplasmic cytochrome c was observed in A2780 cells following cisplatin treatment, whereas dramatic increases were evident in both the CP70 and C30 cell lines. The expression of the mitochondrial factors Bcl-2, Bcl-x, and Bax was determined because each has been implicated in the regulation or release of cytochrome c at the level of the mitochondria. Bcl-2 and Bcl-xL proteins remained relatively unchanged in expression for over 48 h after exposure to cisplatin in the A2780 cell lines. However, within the same time period, expression of Bcl-2 decreased in the CP70- and C30-resistant cell lines, whereas an increase in Bcl-xL expression was observed. Expression of the proapoptotic Bcl-xS protein was observed in only the resistant CP70 and C30 cell lines independent of cisplatin treatment. A change in the expression of Mr 24,000 Bax to a Mr 21,000 isoform was evidenced in the A2780 cells within 48 h of cisplatin treatment and, to a greater extent, in the CP70 and C30 cells, which also expressed a Mr 16,000 Bax variant. Evidence for an alternative apoptotic pathway in A2780 cells was obtained by demonstrating increased FADD expression in response to cisplatin treatment. These results support a model in which cisplatin-induced programmed cell death in the cisplatin-sensitive A2780 and -resistant CP70 and C30 cells proceeds via caspase-3-independent and -dependent pathways, respectively.

Interactions of mammalian proteins with cisplatin-damaged DNA.

We have undertaken the systematic isolation and characterization of mammalian proteins which display an affinity for cisplatin-damaged DNA. Fractionation of human cell extracts has led to the identification of two classes of proteins. The first includes proteins that bind duplex DNA in the absence of cisplatin damage and retain their affinity for DNA in the presence of cisplatin-DNA adducts. The DNA-dependent protein kinase (DNA-PK) falls into this class. The inhibition of DNA-PK phosphorylation activity by cisplatin-damaged DNA has led to the hypothesis that cisplatin sensitization of mammalian cells to ionizing radiation may be mediated by DNA-PK. The second class of proteins identified are those which display a high relative affinity for cisplatin-damaged DNA and a low affinity for undamaged duplex DNA. Proteins that fall into this class include high mobility group 1 protein (HMG-1), replication protein A (RPA) and xeroderma pigmentosum group A protein (XPA). Each protein has been isolated and purified in the lab. The interaction of each protein with cisplatin-damaged DNA has been assessed in electrophoretic mobility shift assays. A series of DNA binding experiments suggests that RPA binds duplex DNA via denaturation and subsequent preferential binding to the undamaged DNA strand of the partial duplex. DNA substrates prepared with photo-reactive base analogs on either the damaged or undamaged DNA strand have also been employed to investigate the mechanism and specific protein-DNA interactions that occur as each protein binds to cisplatin-damaged DNA. Results suggest both damage and strand specificity for RPA and XPA binding cisplatin-damaged DNA.

Induction of apoptosis in cisplatin-sensitive and -resistant human ovarian cancer cell lines.

In this study, we have assessed the mechanism of cytotoxicity in a series of cisplatin-sensitive and -resistant ovarian carcinoma cells following treatment with equitoxic concentrations of cisplatin. The specific proteolytic degradation and the enzymatic activities of the DNA-dependent protein kinase (DNA-PK) were assessed in the cisplatin-sensitive A2780 cell line and two resistant derivative cell lines, CP70 and C30. Forty-eight h following cisplatin treatment, unattached, apoptotic A2780 cells demonstrated a 20-30% decrease in DNA-PK phosphorylation activity. The resistant CP70 and C30 cell lines showed greater decreases in activity approaching 80 and 90%, respectively. The decreases in kinase activity were attributed to proteolytic degradation of the catalytic subunit of DNA-PK (DNA-PKcs). The extent of degradation mimicked the loss of DNA-PK activity, with the resistant cell lines showing the greatest portion of degraded DNA-PKcs. At the same time point, the ability of the DNA-PK Ku subunits to bind DNA was decreased in apoptotic, unattached cells compared to untreated controls, with the decrease in binding activity being attributed to decreased expression of the Ku subunits. In addition to DNA-PKcs cleavage, specific proteolytic cleavage of the poly(ADP-ribose)polymerase and generation of nucleosome-length DNA ladders was observed in all cell lines following cisplatin treatment. These data suggest that cell death via the accumulation of cisplatin-damaged DNA occurs via apoptosis in both the cisplatin-resistant and -sensitive ovarian cancer cells.

Mechanism of DNA-dependent protein kinase inhibition by cis-diamminedichloroplatinum(II)-damaged DNA.

We have determined the mechanism of DNA-dependent protein kinase (DNA-PK) inhibition by cis-diamminedichloroplatinum(II)-(cisplatin-) damaged DNA. We previously have demonstrated that Ku, the DNA binding subunit of DNA-PK, is capable of binding to DNA duplexes globally damaged with cisplatin but was unable to stimulate DNA-PKcs, the catalytic subunit [Turchi & Henkels (1996) J. Biol. Chem. 271, 2992-3000]. In this report we have assessed Ku binding and DNA-PK stimulation using a series of DNA substrates containing single, site-specific d(GpG), d(ApG), and d(GpXpG) intrastrand cisplatin adducts and a substrate with a single interstrand cisplatin adduct. Results demonstrate that Ku binding is marginally decreased by the presence of cisplatin adducts on each substrate. When assayed for the ability to stimulate DNA-PK, each cisplatin-damaged substrate resulted in significantly decreased activity compared to undamaged DNA controls. The degree of inhibition of both Ku binding and kinase activity varied depending on the specific adduct employed. The inhibition of DNA-PK activity by cisplatin-damaged DNA was observed using either a synthetic peptide or human replication protein A as a substrate. Autophosphorylation of the DNA-PKcs and Ku subunits was also inhibited in reactions performed with cisplatin-damaged DNA, demonstrating that increased autophosphorylation of DNA-PKcs does not account for the decreased kinase activity observed with cisplatin-damaged DNA. Equilibrium binding and initial velocity experiments revealed a less than 2-fold increase in the Kd of Ku and the Km of DNA-PK for DNA containing a single 1,2-d(GpG) cisplatin adduct. The mechanism of DNA-PK inhibition by cisplatin-damaged DNA can be attributed to a large decrease in the Vmax and small increase in Km.

Synthesis of the mammalian telomere lagging strand in vitro.

Using a synthetic telomere DNA template and whole cell extracts, we have identified proteins capable of synthesizing the telomere complementary strand. Synthesis of the complementary strand required a DNA template consisting of 10 repeats of the human telomeric sequence d(TTAGGG) and deoxy- and ribonucleosidetriphosphates and was inhibited by neutralizing antibodies to DNA polymerase alpha. No evidence for RNA-independent synthesis of the lagging strand was observed, suggesting that a stable DNA secondary structure capable of priming the lagging strand is unlikely. Purified DNA polymerase alpha/primase was capable of catalyzing synthesis of the lagging strand with the same requirements as those observed in crude cell extracts. A ladder of products was observed with an interval of six bases, suggesting a unique RNA priming site and site-specific pausing or dissociation of polymerase alpha on the d(TTAGGG)10 template. Removal of the RNA primers was observed upon the addition of purified RNase HI. By varying the input rNTP, the RNA priming site was determined to be opposite the 3' thymidine nucleotide generating a five-base RNA primer with the sequence 5'-AACCC. The addition of UTP did not increase the efficiency of priming and extension, suggesting that the five-base RNA primer is sufficient for extension with dNTPs by DNA polymerase alpha. This represents the first experimental evidence for RNA priming and DNA extension as the mechanism of mammalian telomeric lagging strand replication.

High-mobility group 1 protein inhibits helicase catalyzed displacement of cisplatin-damaged DNA.

We have determined the effect of HMG-1 bound to cisplatin-damaged DNA on the activities of calf helicase E. DNase I protection analysis demonstrated HMG-1 bound a cisplatin-damaged 24 base oligonucleotide annealed to M13mp18. Exonuclease digestion experiments revealed that greater than 90% of the DNA substrates contained a single site specific cisplatin adduct and, maximally, 65% of the substrates were bound by HMG-1. Helicase E catalyzed displacement of the cisplatin-damaged DNA oligonucleotide was inhibited by HMG-1 in a concentration-dependent manner. Time course experiments revealed a decreased rate of displacement in reactions containing HMG-1. The maximum inhibition observed was 55% and taking into account that only 65% of the substrates had HMG-1 bound, approximately 85% inhibition was observed on platinated DNA substrates containing HMG-1. Inhibition of helicase activity was proportional to the amount of substrate bound by HMG-1 based on the displacement and exonuclease assays at varying HMG-1 concentrations. The ability of helicase E to displace an undamaged DNA oligonucleotide from a cisplatin-damaged DNA template was also inhibited by HMG-1. Interestingly, HMG-1 had no effect on the rate of DNA-dependent ATP hydrolysis catalyzed by helicase E on the same DNA substrate. The inhibition of helicase activity by HMG-1 binding cisplatin-damaged DNA further supports a role for HMG-1 inhibiting DNA repair which may contribute to cellular sensitivity to cisplatin.

Cisplatin-DNA binding specificity of calf high-mobility group 1 protein.

We have identified a series of proteins with an affinity for cisplatin -damaged DNA using damaged DNA affinity chromatography. We have purified one of these proteins to homogeneity on the basis of a mobility shift assay detecting binding to cisplatin-damaged DNA. The protein was identified as high-mobility group 1 protein (HMG-1) by N-terminal protein sequence analysis. Analysis of a variety of DNA structures revealed that fully duplex DNAs were the best substrates for HMG-1 binding, while partial duplexes were less avidly bound. The decreased levels of binding are attributed to the length of the duplex region of the DNA substrates. A 3-fold increase in binding was observed when a cisplatin-damaged DNA substrate containing a single break in the phosphodiester backbone was joined by DNA ligase. The strict DNA size dependence of binding was also assessed, and a 10-fold increase in binding was observed when the length of the DNA duplex was increased from 44 to 180 base pairs (bp) at the same level of cisplatin damage. HMG-1 binding also was correlated with the degree of cisplatin-DNA damage, suggesting a higher affinity for DNA containing multiple cisplatin adducts. Nuclease degradation of the cisplatin-damaged DNA demonstrated that at the lowest levels of cisplatin damage all of the substrates contained at least one cisplatin adduct. The potential role of HMG-1 in the repair of cisplatin-DNA adducts is discussed.