Novel anti-apoptotic effect of Bcl-2: prevention of polyamine depletion-induced cell death

The spermine analogue N(1),N(11)-diethylnorspermine (DENSPM) efficiently depletes the polyamine pools in the breast cancer cell line L56Br-C1 and induces apoptotic cell death via the mitochondrial pathway. In this study, we have over-expressed the anti-apoptotic protein Bcl-2 in L56Br-C1 cells and investigated the effect of DENSPM treatment. DENSPM-induced cell death was significantly reduced in Bcl-2 over-expressing cells. Bcl-2 over-expression reduced DENSPM-induced release of the pro-apoptotic proteins AIF, cytochrome c, and Smac/DIABLO from the mitochondria. Bcl-2 over-expression reduced the DENSPM-induced activation of caspase-3. Bcl-2 over-expression also prevented DENSPM-induced Bax cleavage and reduction of Bcl-X(L) and survivin levels. The DENSPM-induced activation of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase was reduced by Bcl-2 over-expression, partly preventing polyamine depletion. Thus, Bcl-2 over-expression prevented a number of DENSPM-induced apoptotic effects.     

The retinoblastoma protein (Rb) as an anti-apoptotic factor: expression of Rb is required for the anti-apoptotic function of BAG-1 protein in colorectal tumour cells

Although the retinoblastoma-susceptibility gene RB1 is inactivated in a wide range of human tumours, in colorectal cancer, the retinoblastoma protein (Rb) function is often preserved and the RB locus even amplified. Importantly, we have previously shown that Rb interacts with the anti-apoptotic Bcl-2 associated athanogene 1 (BAG-1) protein, which is highly expressed in colorectal carcinogenesis. Here we show for the first time that Rb expression is critical for BAG-1 anti-apoptotic activity in colorectal tumour cells. We demonstrate that Rb expression not only increases the nuclear localisation of the anti-apoptotic BAG-1 protein, but that expression of Rb is required for inhibition of apoptosis by BAG-1 both in a γ-irradiated Saos-2 osteosarcoma cell line and colorectal adenoma and carcinoma cell lines. Further, consistent with the fact that nuclear BAG-1 has previously been shown to promote cell survival through increasing nuclear factor (NF)-κB activity, we demonstrate that the ability of BAG-1 to promote NF-κB activity is significantly inhibited by repression of Rb expression. Taken together, data presented suggest a novel function for Rb, promoting cell survival through regulating the function of BAG-1. As BAG-1 is highly expressed in the majority of colorectal tumours, targeting the Rb–BAG-1 complex to promote apoptosis has exciting potential for future therapeutic development.     

Novel galactosylated polyamine bolaamphiphiles for gene delivery

We describe the synthesis of a series of alpha-galacto-omega-polyamine double-chain bolaamphiphiles (Gal-CL) and report on the gene transfer mediated with lipoplexes they form either when used in conjunction with DOPE or with pcTG90:DOPE. Lipofection was investigated with human HepG2 and murine BNL-CL2 hepatocytes expressing the asialoglycoprotein (ASGP) receptor which displays a high affinity for galactosyl residues, and with A549 cells which do not express ASGP. Our results show that cationic N/P = 5 and 2.5 Gal-CL lipoplexes constitute very efficient nonspecific gene transfer systems. Lipofection experiments performed in the presence of asialofetuin (a high affinity ligand of ASGP) led us to evidence also the involvement of a specific receptor-mediated endocytosis pathway for the transfection of the ASGP(+) HepG2 or BNL-CL2 hepatocytes with some Gal-CL formulations. This work suggests that targetable lipopolyamines presenting a single galactose residue appear as promising synthetic vectors for specific gene delivery to ASGP(+) cells.     

A role for the retinoblastoma protein as a regulator of mouse osteoblast cell adhesion: implications for osteogenesis and osteosarcoma formation

The retinoblastoma protein (pRb) is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis.     

Heat-shock proteins attenuate SERCA inactivation by the anti-apoptotic protein Bcl-2: possible implications for the ER Ca2+-mediated apoptosis

We have demonstrated previously that Bcl-2 and Bcl-2Δ21, a C-terminally truncated Bcl-2 sequence, inactivate SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase) 1 in isolated SR (sarcoplasmic reticulum), accompanied by a translocation from CRDs (caveolae-related domains) of the SR. In the present study, we obtained evidence for the interaction of Bcl-2 with SERCA2b in C2C12 myoblasts and HEK (human embryonic kidney)-293 cells. Bcl-2 and SERCA2b co-immunoprecipitated from lysate and microsomal fractions of Bcl-2-overexpressing cells. However, Bcl-2 overexpression resulted only in a slight translocation from the CRDs and no significant SERCA inactivation. In isolated HEK-293 cell microsomes, incubation with Bcl-2Δ21 afforded SERCA2b inactivation and some translocation. HSP (heat-shock protein) 70, HSP90, HSP27 and α-crystallin attenuated Bcl-2Δ21-dependent SERCA2b inactivation. An in vitro mechanistic study with the SERCA1 isoform shows that HSP70 (i) protects SERCA1 from the inactivation by Bcl-2Δ21, (ii) inhibits SERCA1 translocation from CRD fractions, and (iii) prevents the Bcl-2Δ21-dependent loss of FITC labelling. Our data demonstrate that the mechanism of SERCA inactivation by Bcl-2 established in vitro for the SERCA1 isoform can be extended to the main housekeeping SERCA2b isoform, and that functional interactions of SERCA2b and Bcl-2 in the cell may be modulated by HSP70 and other chaperones and stress-regulated proteins.     

Inhibition of acetylpolyamine and spermine oxidases by the polyamine analogue chlorhexidine

Acetylpolyamine and spermine oxidases are involved in the catabolism of polyamines. The discovery of selective inhibitors of these enzymes represents an important tool for the development of novel anti-neoplastic drugs. Here, a comparative study on acetylpolyamine and spermine oxidases inhibition by the polyamine analogue chlorhexidine is reported. Chlorhexidine is an antiseptic diamide, commonly used as a bactericidal and bacteriostatic agent. Docking simulations indicate that chlorhexidine binding to these enzymes is compatible with the stereochemical properties of both acetylpolyamine oxidase and spermine oxidase active sites. In fact, chlorhexidine is predicted to establish several polar and hydrophobic interactions with the active site residues of both enzymes, with binding energy values ranging from ?7.6 to ?10.6 kcal/mol. In agreement with this hypothesis, inhibition studies indicate that chlorhexidine behaves as a strong competitive inhibitor of both enzymes, values of K_i being 0.10 μM and 0.55 μM for acetylpolyamine oxidase and spermine oxidase, respectively.     

Increased breast cancer cell toxicity by palladination of the polyamine analogue N 1,N 11-bis(ethyl)norspermine

Breast cancer is one of the most common malignant tumor forms among women and many women succumb to their disease. Thus, new anticancer agents that can efficiently improve patient survival are of the utmost importance. In this study, the effects of the polyamine analogues N ¹,N ¹¹-bis(ethyl)norspermine (BENSpm) and N ¹-cyclo-propylmethyl-N ¹¹-ethylnorspermine (CPENSpm) and the synthesized dinuclear complexes Pd₂BENSpm (Pd-BENSpm), Pt₂CPENSpm (Pt-CPENSpm) and Pd₂Spm (Pd-Spm) were investigated in normal-like breast epithelial MCF-10A cells and the breast cancer cell lines JIMT-1 and L56BR-C1. The overall data show that palladination of BENSpm resulted in enhanced cytotoxicity, in contrast to platination of CPENSpm that reduced cytotoxicity, which might be explained by differences in the cellular uptake of Pd-BENSpm and Pt-CPENSpm. BENSpm and Pd-BENSpm treatment reduced the CD44⁺CD24^? putative cancer stem cell population, evaluated by flow cytometry. Furthermore, Pd-BENSpm was the most efficient compound regarding induction of DNA damage and decrease in colony formation in soft agar. Pt-CPENSpm and Pd-Spm, on the other hand, were shown to be the least toxic compounds of all tested. Pd-Spm efficiently reduced the cellular glutathione levels, which probably was a consequence of its metabolic inactivation by conjugation to this endogenous thiol. The normal-like cells were found to be less sensitive to the agents than the breast cancer cells. Our findings show that Pd-BENSpm exhibits promising anticancer effects which render it suitable for further optimization to develop a new metal-based chemotherapeutic drug for breast cancer treatment.     

Evidence for a mitochondrial localization of the retinoblastoma protein

Background  

The retinoblastoma protein (Rb) plays a central role in the regulation of cell cycle, differentiation and apoptosis. In cancer cells, ablation of Rb function or its pathway is a consequence of genetic inactivation, viral oncoprotein binding or deregulated hyperphosphorylation. Some recent data suggest that Rb relocation could also account for the regulation of its tumor suppressor activity, as is the case for other tumor suppressor proteins, such as p53.     

Subcellular localization of the retinoblastoma protein

The subcellular localization of the retinoblastoma (RB) protein has been studied in primate cell lines by immunofluorescence staining using different monoclonal and polyclonal antibodies. The protein appeared as granules of heterogeneous size over the interphase nuclei. Computer assisted digital overlap analysis indicated that it was predominantly localized in euchromatic areas with low DNA density. The largest RB positive grains lined up on the heterochromatin/euchromatin boundary. During mitosis, the RB protein dissociated from the condensing chromosomes. It was dispersed throughout the cytoplasm during metaphase and anaphase, and it reassociated with the decondensing chromatin during telophase. A new monoclonal antibody, designated aRB1C1, was raised against a bacterial TrpE/human retinoblastoma protein. It specifically recognized the nonphosphorylated and differentially phosphorylated forms of the RB protein in immunoprecipitation experiments. A collection of RB expressing cell lines gave a positive staining reaction with the antibody, whereas the RB negative Weri-RB-27 retinoblastoma and OHS osteosarcoma cells failed to react. Wild-type RB complementary DNA was introduced into Weri-RB-27 by retrovirus mediated gene transfer. Similar experiments were performed with the DU145 prostatic carcinoma cell line that expresses a mutant RB protein. Reconstituted cells of both lines expressed the normal size RB protein and gave a positive immunofluorescence reaction with the aRB1C1 and other anti-RB antibodies. The new monoclonal antibody, however, showed cell type dependent differences of the staining pattern compared to other anti-RB antibodies, suggesting differentiation dependent accessibility to its epitope.     

Hereditary retinoblastoma: Lack of maternal effect

Summary Analysis of two-generation data from published pedigrees with familial cases of retinoblastoma showed that, while the proportion of bilateral cases among affected children varied consistently with expressivity of the parents who transmitted the gene, the proportion did not differ with the sex of the parents. These findings support the view that inherited host resistance to the development of hereditary retinoblastoma is determined by modifying genes, and not influenced by transplacental maternal factors, and that the resistance is mainly directed to the induction (promotion) phase. There was a significant deficit of bilaterally affected mothers relative to bilaterally affected fathers, which could not be accounted for by ascertainment bias or by differential survival or fertility between sexes. It is suggested that female patients who survived bilateral retinoblastoma had much less chance of marriage as compared to male survivors. Examination of pedigrees with multiple cases of retinoblastoma, transmitted via unaffected carriers, revealed no sign of cytoplasmic inheritance associated with a vertically transmitting virus.     

Rapamycin induces the anti-apoptotic protein survivin in neuroblastoma

Neuroblastoma is the most common solid tumor of infancy, accounting for 15% of all cancer cell deaths in children. Expression of the anti-apoptotic protein survivin in these tumors correlates with poor prognostic features and resistance to therapy. The mammalian target of rapamycin (mTOR) protein is being explored as a potential therapeutic target in patients with this disease. The objective of this study was to test the hypothesis that rapamycin regulates survivin expression and function in neuroblastoma cells. To explore this hypothesis, we treated two different neuroblastoma lines (NB7, NB8) and a well-characterized control lung cancer cell line, A549, with varying doses of rapamycin (0.1-10μM) for serial time points (2-48 hours). RNA and protein expression levels were then evaluated by quantitative RT-PCR and western blotting, respectively. Cell proliferation and apoptosis were assayed by WST-1 and Annexin V. The results showed a rapamycin-dependent increase in survivin mRNA and protein levels in the neuroblastoma cell lines in a dose- and time-dependent fashion, while a decrease in these levels was observed in control cells. Rapamycin inhibited cell proliferation in both A549 and neuroblastoma cells however neuroblastoma cells had less apoptosis than A549 cells (9% vs. 20%). In summary, our results indicate that rapamycin induces expression of the anti-apoptotic protein survivin in neuroblastoma cells which may protect these cells from programmed cell death. Induction of survivin by rapamycin could therefore be a potential mechanism of neuroblastoma tumor cell resistance and rapamycin may not be an effective therapeutic agent for these tumors.     

In vitro stimulatory effect of anti-apoptotic seminal vesicle protein 4 on purified peroxidase enzymes

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 mum native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme-electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.     

NMR studies of the anti-apoptotic protein Bcl-xL in micelles

The Bcl-2 family of proteins play a pivotal role in the regulation of programmed cell death. One of the postulated mechanisms for the function of these proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporated into small detergent micelles. This form of Bcl-x(L) lacks the loop (residues 49-88) between helix 1 and helix 2 and the putative C-terminal transmembrane helix (residues 214-237). Below the critical micelle concentration (CMC), Bcl-x(L) binds detergents in the hydrophobic groove that binds to pro-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary structure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous solution, the structure of the protein when dissolved in dodecylphosphocholine (DPC) micelles consists of several alpha-helices separated by loops. However, the length and position of the individual helices of Bcl-x(L) in micelles differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and limited proteolysis. In addition, the mobility of the micelle-bound form of Bcl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and location of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dynamic structure in micelles, with helices 1 and 6 and possibly helix 5 partially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.     

Frequency of somatic and germ-line mosaicism in retinoblastoma: implications for genetic counseling.

Although mosaicism can have important implications for genetic counseling of families with hereditary disorders, information regarding the incidence of mosaicism is available for only a few genetic diseases. Here we describe an evaluation of 156 families with retinoblastoma; the initial oncogenic mutation in the retinoblastoma gene had been identified in these families. In 15 ( approximately 10%) families, we were able to document mosaicism for the initial mutation in the retinoblastoma gene, either in the proband or in one of the proband's parents. The true incidence of mosaicism in this group of 156 families is probably higher than our findings indicate; in some additional families beyond the 15 we identified, mosaicism was likely but could not be proven, because somatic or germ-line DNA from key family members was unavailable. Germ-line DNA from two mosaic fathers was analyzed: in one of these, the mutation was detected in both sperm and leukocyte DNA; in the other, the mutation was detected only in sperm DNA. Our data suggest that mosaicism is more common than is generally appreciated, especially in disorders such as retinoblastoma, in which a high proportion of cases represent new mutations. The possibility of mosaicism should always be considered during the genetic counseling of newly identified families with retinoblastoma. As demonstrated here, genetic tests of germ-line DNA can provide valuable information that is not available through analysis of somatic (leukocyte) DNA.