Inhibition of focal adhesion kinase by antisense oligonucleotides enhances the sensitivity of breast cancer cells to camptothecins

This study shows a strong association between cell attachment to substratum and activation of beta 1-integrin-signaling with resistance to the camptothecin derivative topotecan (TPT) in breast cancer cells. We propose a mechanistic-driven approach to sensitize the cells to camptothecins. ZR-75-1 anchorage-dependent breast cancer cell line, its derivative 9D3S suspension cells (9D3S-S), and 9D3S cells attached to fibronectin-coated plates (9D3S-A) were treated with TPT (1 microM) or CPT-11 (40 microM) for 48 h. Programmed cell death (PCD), as shown by poly(ADP-ribose) polymerase (PARP), pro-caspase-3 and pro-caspase-9 cleavage, was observed in 9D3S-S cells but not in ZR-75-1 or 9D3S-A cells. Because p125 focal adhesion kinase (FAK) is a transducer in the beta 1-integrin signaling pathway, it is essential to cell adhesion and it is overexpressed in metastatic breast cancer, we hypothesized that attenuation of FAK might enhance the sensitivity of breast cancer cells to camptothecins. Moreover, inhibition of FAK gene expression by a phosphorothioated antisense oligodeoxynucleotide targeting the portion of the gene encoding amino acids 262-268, increased the sensitivity of ZR-75-1, MDA-MB-231 and MCF7 breast cancer cells to treatment with TPT or CPT-11. (AU)

Inhibition of focal adhesion kinase by antisense oligonucleotides enhances the sensitivity of breast cancer cells to camptothecins

This study shows a strong association between cell attachment to substratum and activation of beta 1-integrin-signaling with resistance to the camptothecin derivative topotecan (TPT) in breast cancer cells. We propose a mechanistic-driven approach to sensitize the cells to camptothecins. ZR-75-1 anchorage-dependent breast cancer cell line, its derivative 9D3S suspension cells (9D3S-S), and 9D3S cells attached to fibronectin-coated plates (9D3S-A) were treated with TPT (1 microM) or CPT-11 (40 microM) for 48 h. Programmed cell death (PCD), as shown by poly(ADP-ribose) polymerase (PARP), pro-caspase-3 and pro-caspase-9 cleavage, was observed in 9D3S-S cells but not in ZR-75-1 or 9D3S-A cells. Because p125 focal adhesion kinase (FAK) is a transducer in the beta 1-integrin signaling pathway, it is essential to cell adhesion and it is overexpressed in metastatic breast cancer, we hypothesized that attenuation of FAK might enhance the sensitivity of breast cancer cells to camptothecins. Moreover, inhibition of FAK gene expression by a phosphorothioated antisense oligodeoxynucleotide targeting the portion of the gene encoding amino acids 262-268, increased the sensitivity of ZR-75-1, MDA-MB-231 and MCF7 breast cancer cells to treatment with TPT or CPT-11.

Potential involvement of C(3) complement factor in amphibian fertilization.

We have assessed the potential involvement of C(3), the third complement factor, and its receptor in Bufo arenarum fertilization. We show that a polyclonal antibody against a B. arenarum C(3)-like factor (C(3)Ba) reacts specifically with components of the extracellular matrix (ECM) of coelomic eggs and the cell membrane of uterine eggs. Interestingly, we have identified a 163 kD protein immunoreactive with a monoclonal antibody against the CD11b alpha chain of the human C(3) receptor on the cell membrane of the animal pole of uterine eggs, the site of entry of the sperm, but not in coelomic eggs (CR3Ba). Treatment of coelomic eggs with a pars recta oviductal-like protease, trypsin, induced the translocation of C(3)Ba from the ECM to the cell membrane. Furthermore, inhibition of CR3Ba by trypan blue, as well as inhibition of C(3)Ba by anti-C(3)Ba on uterine eggs impaired fertilization, whereas identical treatment on sperm cells did not alter percentage fertilization. Our results suggest, (A) that changes in the localization of C(3)Ba from the ECM to the cell membrane may be triggered by trypsin-like proteases during passage of eggs through the oviduct; and (B) that C(3)Ba/CR3Ba may be involved in B. arenarum fertilization.

Photodynamic therapy with the phthalocyanine photosensitizer Pc 4 of SW480 human colon cancer xenografts in athymic mice.

Photodynamic therapy (PDT) using the silicon phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N-(CH3)2] is an oxidative stress associated with induction of apoptosis in various cell types. We assessed the effectiveness of Pc 4-PDT on SW480 colon cancer xenografts grown in athymic nude mice. Animals bearing xenografts were treated with 1 mg/kg body weight Pc 4 and 48 h later were irradiated with 150 J/cm2 672-nm light from a diode laser delivered at 150 mW/cm2. Biochemical studies were performed in xenografts resected at various time points up to 26 h after Pc 4-PDT treatment, whereas tumor size was evaluated over a 4-week period in parallel experiments. In the tumors resected for biochemical studies, apoptosis was visualized by activation of caspase-9 and caspase-3 and a gradual increase in the cleavage of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) to a maximum of approximately 60% of the total PARP present at approximately 26 h. At that time all Pc 4-PDT-treated tumors had regressed significantly. Two signaling responses that have previously been shown to be associated with Pc 4-PDT-induced apoptosis in cultured cells, p38 mitogen-activated protein kinase and p21/WAF1/Cip1, were examined. A marked increase in phosphorylation of p38 was observed within 1 h after Pc 4-PDT without changes in levels of the p38 protein. Levels of p21 were not altered in the xenografts in correspondence with the presence of mutant p53 in SW480 cells. Evaluation of tumor size showed that tumor growth resumed after a delay of 9-15 days. Our results suggest that: (a) Pc 4-PDT is effective in the treatment of SW480 human colon cancer xenografts independent of p53 status; (b) PARP cleavage may be mediated by caspase-9 and caspase-3 activation in the Pc 4-PDT-treated tumors; and (c) p38 phosphorylation may be a trigger of apoptosis in response to PDT in vivo in this tumor model.

A simple procedure for isolation of Bufo arenarum C3 complement fraction and preparation of antiserum.

Because of the need for antibodies in our studies involving the third component of complement in Bufo arenarum, we performed a simple procedure to purify C3 from B. arenarum serum to use as antigen in the preparation of the antiserum. The strategy was based on the well-known ability of C3 to bind to zymosan (Zy), a yeast cell wall extract comprised of polysaccharides. The Zy-bound fraction showed cross reactivity with a commercial antibody to human C3 as well as a similar electrophoretic profile (SDS-PAGE) to C3 from other species. The Zy-C3 complex resulting from binding Zy to B. arenarum serum was injected into rabbits and the antiserum against this C3-like fraction was purified by protein A-Sepharose chromatography. The purified C3 antibody was found to be suitable for immunochemical studies.

Pharmacologic disruption of base excision repair sensitizes mismatch repair-deficient and -proficient colon cancer cells to methylating agents.

Previously we showed that a mismatch repair (MMR)-deficient cell line, HCT116 (hMLH1 mut), unlike a MMR wild-type cell line, SW480, was more resistant to the therapeutic methylating agent, temozolomide (TMZ), because the MMR complex fails to recognize TMZ-induced O6-methylguanine DNA adduct mispairings with thymine that arise after replication. TMZ also produces N7-methylguanine and N3-methyladenine adducts that are processed efficiently by the base excision repair (BER) system. After removal of the methylated base by methylpurine glycosylase, which creates the abasic or apurinic-apyrimidinic (AP) site, the phosphodiester bond is hydrolyzed immediately by AP endonuclease, initiating the repair of the AP site. Methoxyamine (MX) reacts with the abasic site and prevents AP endonuclease cleavage, disrupting DNA repair. MX potentiated the cytotoxic effect of TMZ with a dose modification factor (DMF) of 2.3+/-0.12 in SW480 and 3.1+/-0.16 in HCT116. When combined with O6-benzylguanine (BG), MX and TMZ dramatically increased TMZ cytotoxicity (65.8-fold) in SW480, whereas no additive effect was seen in HCT116. This suggests that N7-methylguanine and N3-methyladenine adducts are cytotoxic lesions in MMR-deficient and wild-type cells when BER is interrupted. Because poly(ADP-ribose) polymerase (PARP) aids in processing of DNA strand breaks induced during MMR and BER, we asked whether PARP inhibitors would also affect BER-mediated cell killing. We found that PARP inhibitors PD128763, 3-aminobenzimide, and 6-aminonicotinamide increased the sensitivity to TMZ in both HCT116 MMR-deficient cells and SW480 MMR wild-type cells. In HCT116 cells, PD128763 remarkably decreased resistance to TMZ, with a DMF of 4.7+/-0.2. However, the combination of PD128763, BG, and TMZ had no greater effect, indicating that persistent O6-methylguanine had no effect on cytotoxicity. In SW480, the DMF for TMZ cytotoxicity was 3.1+/-0.12 with addition of PD128763 and 36 with addition of PD128763 and BG. Synergy analysis by median effect plots indicated a high degree of synergy between TMZ and MX or PD128763. In contrast, 1,3-bis(2-chloroethyl)-1-nitrosourea combined with either MX or PD128763 showed little if any potentiation observed in the absence of BG in either cell line, suggesting that BER pathway has little impact on cytotoxic processing of 1,3-bis(2-chloroethyl)-1-nitrosourea-induced adducts. These studies indicate that targeting BER with MX or PARP inhibitors enhances the cytotoxicity of methylating agents, even in MMR-deficient cells.

Detection of poly(ADP-ribose) polymerase cleavage in response to treatment with topoisomerase I inhibitors: a potential surrogate end point to assess treatment effectiveness.

Cleavage of poly(ADP-ribose) polymerase (PARP) by caspases is a prominent characteristic of apoptosis or programmed cell death shown to be induced by topoisomerase (Topo) inhibitors. Because Topo I inhibitors have been shown to be effective in the treatment of some patients with colon cancer, we considered the possibility of using PARP cleavage as an early predictor of responsiveness to this class of agents. We show cleavage of PARP in response to treatment with Topo I inhibitors in colon cancer both in vitro and in vivo: (a) in vitro in SW480, HCT116, VACO5, VACO6, VACO8, VACO411, VACO425, and VACO451 human colon cancer cell lines treated with topotecan (TPT) or CPT-11; (b) in vivo in SW480, VACO451, and VRC5 colon cancer xenografts grown in athymic mice treated with TPT or CPT-11; and (c) in vivo in colon cancer samples from patients undergoing a Phase II clinical trial with CPT-11. Our results show a strong correlation between percentage of PARP cleavage and percentage of acridine orange-positive cells in colon cancer cell lines treated with 0.1 microM TPT for 24 and 48 h, confirming that PARP cleavage is a useful marker for programmed cell death in colon cancer cell lines. Results from experiments performed on colon cancer xenografts also show an association between PARP cleavage and response to treatment with TPT or CPT-11. The increase of PARP cleavage in xenografts and in clinical samples corresponding to treatment with Topo I inhibitors suggests that this procedure may have early predictive value to assess effectiveness of treatment. These results provide the basis for determining the validity of using PARP cleavage as an early marker of chemotherapeutic effectiveness in human samples.

Apoptosis induction by the glucocorticoid hormone dexamethasone and the calcium-ATPase inhibitor thapsigargin involves Bc1-2 regulated caspase activation.

The requirement for caspases (ICE-like proteases) were investigated in mediating apoptosis of WEHI7.2 mouse lymphoma cells in response to two death inducers with different mechanisms of action, the glucocorticoid hormone dexamethasone (DX) and the calcium-ATPase inhibitor thapsigargin (TG). Apoptosis induction by these agents followed different kinetics, and was closely correlated with in vivo activation of caspase-3 (CPP32/Yama/Apopain) and cleavage of the caspase target protein poly(ADP-ribose) polymerase (PARP). Caspase activation and PARP cleavage were inhibited by Bcl-2 overexpression. Cell extracts from DX- and TG-treated cells cleaved the in vitro synthesized baculovirus p35 ICE-like protease target, producing 25 and 10 kDa fragments. p35 cleavage was inhibited by mutating the active site aspartic acid to alanine, and by a panel of protease inhibitors that inhibit caspase-3-like proteases, including iodoacetamide, N-ethylmaleimide, and Ac-DEVD-cho. Treatment of cells in vivo with two cell permeant peptide fluoromethylketone inhibitors of caspase activity, Z-VAD-fmk and Z-DEVD-fmk, inhibited DX- and TG-induced apoptotic nuclear changes and maintained plasma membrane integrity, whereas the cathepsin inhibitor, Z-FA-fmk, and two calpain inhibitors failed to inhibit apoptosis. An unexpected observation was that due to the delayed time course of DX-induced apoptosis, optimal preservation of plasma membrane integrity was achieved by adding caspase inhibitors beginning 8 h after DX addition. In summary, the findings indicate that two diverse apoptosis-inducing signals converge into a common Bcl-2-regulated pathway that leads to caspase activation and apoptosis.

New isolation technique to study apoptosis in human intestinal epithelial cells.

Intestinal epithelial cells derive from stem cells at the base of the crypt and migrate along the crypt-lumen axis. Their life is terminated as they reach the luminal surface where they detach and are shed. Intestinal epithelial cells show evidence of apoptosis in the region of shedding, and cell death is thought to resemble a form of apoptosis called detachment-induced cell death, or anoikis. Human intestinal epithelial cells die rapidly in vitro due to loss of anchorage during isolation, making primary culture of these cells a goal that has not yet been reached. However, the molecular mechanisms underlying this process of anoikis are largely unknown. In this study, a novel protocol for the rapid, temperature-controlled isolation of highly purified human colonic epithelial cells from surgical specimens is described. Using this method, early molecular events of anoikis in nontransformed epithelial cells were studied. Intestinal epithelial cells were isolated at the beginning of the apoptotic cascade, before the activation of caspase 3 family members and cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. Elucidating the molecular mechanisms of detachment-induced cell death may facilitate the establishment of long-term primary cultures of human intestinal epithelial cells and enhance our understanding of homeostasis in the intestinal epithelium.

Involvement of blood serum in amphibian fertilization

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization

Involvement of blood serum in amphibian fertilization

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization

Protease activation and cleavage of poly(ADP-ribose) polymerase: an integral part of apoptosis in response to photodynamic treatment.

Apoptosis induced by numerous cancer chemotherapeutic and other toxic agents has been shown to proceed through a cascade of proteases, now termed caspases, culminating in cleavage of a set of proteins. The ability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to activate cellular caspases has been assessed during the rapid apoptosis in murine lymphoma L5178Y-R cells. Cells were exposed to combinations of Pc 4 and activating red light that result in > or =90% cell death, as judged by a clonogenic assay. The rate of entry of cells into apoptosis was dose dependent. For 0.5 microM Pc 4 and either 2.1 or 3 kJ/m2, which kill 90 or 99.9% of the cells, oligonucleosomal fragmentation was visible on agarose gels as early as 60 or 30 min after PDT, respectively. To assess caspase activation, cells were harvested at various times after PDT, and cell proteins were subjected to electrophoresis and Western blot analysis, using an antibody to poly(ADP-ribose) polymerase (PARP). The cleavage of the normally Mr 116,000 PARP into fragments of Mr approximately 90,000 was observed at approximately the same time as the earliest DNA fragmentation. An antibody to the polymer, poly(ADP-ribose), did not recognize the Mr approximately 90,000 PARP cleavage products, in contrast to the parent enzyme. This analysis also revealed that levels of a poly(ADP-ribosylated) Mr 100,000 protein, tentatively identified as topoisomerase I, were maintained in cells after PARP was fully cleaved. Caspase-3 (and/or caspase-7) activity, as measured in cell lysates with the fluorogenic substrate DEVD-AMC, was elevated almost immediately after PDT. The cell-permeable, irreversible caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoro-methylketone, inhibited PDT-induced apoptosis and PARP cleavage, whereas the inactive peptide analogue, benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone, was without effect. The results indicate that PDT-induced apoptosis is mediated by activation of caspase-3 and/or other similar caspases.

Involvement of blood serum in amphibian fertilization.

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization.

Factors affecting topotecan-induced programmed cell death: adhesion protects cells from apoptosis and impairs cleavage of poly(ADP-ribose)polymerase.

We have evaluated the influence of anchorage status together with endogenous levels of bcl-2 family members on the ability of the topoisomerase I inhibitor, topotecan (TPT), to induce programmed cell death (PCD) in human colon, breast, lymphoid, and cervical cancer cell lines. As part of this study, we assessed the use of measuring poly(ADP-ribose) polymerase (PARP) cleavage by Western blot, as an index of apoptosis, relative to measuring chromatin condensation by acridine orange analysis. Our results show a strong correlation between both assays, indicating that PARP cleavage is an accurate method to examine PCD. We have encountered a strong association between cell attachment and sensitivity to TPT-induced PCD. Cells growing attached to flasks appear to be relatively more resistant than suspension-growing cells in spite of endogenous bcl-2, bax, or bcl-x levels. Furthermore, we demonstrate that interference with attachment status alters the sensitivity of cells to TPT-induced PCD. Although cell attachment to ProNectin F confers protection against TPT-induced chromatin condensation and cleavage of PARP, cell detachment by poly(2-hydroxyethyl methacrylate) stimulates TPT-induced PCD and PARP cleavage.

Topotecan increases topoisomerase IIalpha levels and sensitivity to treatment with etoposide in schedule-dependent process.

To elucidate the effect of topoisomerase (Topo) I inhibitors in the modulation of Topo II levels and sensitivity to Topo II-directed drugs, athymic mice bearing SW480 human colon cancer xenografts were treated with simultaneous, subsequent, or distant doses of topotecan and etoposide. This in vivo study demonstrates that simultaneous administration of topotecan and etoposide results in an antagonistic response. In contrast, inhibition of Topo I by topotecan results in a compensatory increase in Topo II alpha levels associated with increasing sensitivity of tumors to subsequent treatment with the Topo II inhibitor etoposide. Furthermore, we show that Topo II alpha levels decline 5 days after the last dose of topotecan, resulting in restoration of the original response of the xenografts to etoposide. Thus, this study emphasizes the critical role of schedule dependency to optimize the effectiveness of combination chemotherapy with Topo I and Topo II inhibitors.

Involvement of NAD-poly(ADP-ribose) metabolism in p53 regulation and its consequences.

We have used two different approaches to study the consequences of NAD/poly(ADP-ribose) deficiency on p53 expression and its activity in V79-derived cell lines. In the first approach, we have used two cell lines that are deficient in poly(ADP-ribose) (pADPR) synthesis because of deficiency in the enzyme poly(ADP-ribose) polymerase (PARP). In a second approach, we have used a cell line that is deficient in NAD/pADPR metabolism due to unavailability of NAD, the substrate for PARP. These NAD/PARP-deficient cell lines exhibit a significant reduction in both baseline p53 expression and its activity compared to their parental V79 cells. Furthermore, etoposide, a topoisomerase II inhibitor that was shown to cause an increase in p53 expression and subsequent apoptosis in V79 cells, failed to produce any significant increase in p53 expression or apoptotic DNA fragmentation in NAD/PARP-deficient cell lines. Thus, our studies suggest that NAD/pADPR synthesis may be involved in the regulation of p53 and its dependent pathways.

Poly(adenosine diphosphoribose) polymerase in peripheral blood leukocytes from normal donors and patients with malignancies.

A two-color flow cytometric technique was developed to analyze poly(ADP-ribose) polymerase (PADPRP) in different individuals as a function of different physiological or pathological conditions and to establish the basis for determining whether enzyme deficiency may predispose to degenerative or malignant disorders. Peripheral blood granulocytes were devoid of enzyme activity, whereas mononuclear cells had variable amounts. PADPRP was highest in B cells, intermediate in T cells, and lowest in monocytes. This pattern of enzyme distribution and relative enzyme content of different types of cells was remarkably constant in normal subjects. In a series of 66 normal donors there was no significant biological variation in enzyme content as a function of age, race, or sex. The mean PADPRP values in peripheral blood mononuclear cells from 81 random patient samples obtained from an ambulatory oncology clinic did not differ significantly from normal subjects. However, groups of patients with breast cancer, lymphocytic malignancies, and esophageal cancer were observed to have below normal levels for peripheral blood mononuclear cell PADPRP.