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Biomedical subjects

R Wolkowicz

Publications and source records attributed to R Wolkowicz.

17 recordsLinked to original sources
PubMed2005

Gene therapy progress and prospects: novel gene therapy approaches for AIDS.

Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV), kills millions worldwide every year. Vaccines against HIV still seem a distant promise. Pharmaceutical treatments exist, but these are not always effective, and there is increasing prevalence of viral strains with multidrug resistance. Highly active antiretroviral therapy (HAART) consists of inhibitors of viral enzymes (reverse transcriptase (RT) and protease). Gene therapy, first introduced as intracellular immunization, may offer hopes for new treatments to be used alone, or in conjunction with, conventional small molecule drugs. Gene therapy approaches against HIV-1, including suicide genes, RNA-based technology, dominant negative viral proteins, intracellular antibodies, intrakines, and peptides, are the subject of this review.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Oligonucleotide-directed site-specific integration of high complexity libraries into ssDNA templates.

We present an approach that generates an oligomer-based library with minimal need for restriction site modification of sequences in the target vector. The technique has the advantage that it can be applied for generating peptide aptamer libraries at sites within proteins without the need for introducing flanking enzyme sites. As an example we present a phagemid retroviral shuttle vector that can be used to achieve stable expression of the library in mammalian cells for the purpose of screening for peptides with desired biological activity.

Base Sequence↗
PubMed2000

Systemic lupus erythematosus in mice, spontaneous and induced, is associated with autoimmunity to the C-terminal domain of p53 that recognizes damaged DNA.

The tumor suppressor molecule p53 features a regulatory domain at the C terminus that recognizes damaged DNA. Since damaged DNA might be involved in activating anti-DNA autoantibodies, we tested whether autoimmunity to the C terminus of p53 might mark murine systemic lupus erythematosus (SLE). We now report that MRL / MpJ-Fas(lpr) mice, which spontaneously develop SLE, produce antibodies both to the C terminus of p53 and to a monoclonal antibody (PAb-421) that binds the p53 C terminus. Anti-idiotypic antibodies to PAb-421 (sampled as monoclonal antibodies) could also bind DNA. Thus, the PAb-421 antibody mimics DNA, and the anti-idiotypic antibody to PAb-421 mimics the p53 DNA-binding site. This mimicry was functional; immunization of BALB / c mice to PAb-421 induced anti-DNA antibodies and antibodies to the C terminus of p53, and most of the mice developed an SLE-like disease. Immunization of C57BL / 6 mice to PAb-421 induced antibodies to p53, but not to its C-terminal domain. The C57BL / 6 mice also did not develop anti-DNA antibodies or the SLE-like disease. Thus, network autoimmunity to the domain of p53 that recognizes damaged DNA can be a pathogenic feature in SLE in genetically susceptible strains of mice.

Animals↗
PubMed1999

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery.

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature-sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild-type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.

Animals↗
PubMed1999

COOH-terminal domain of p53 modulates p53-mediated transcriptional transactivation, cell growth, and apoptosis.

The tumor suppressor protein p53 contributes to the control of cell cycle checkpoints and stress-induced apoptosis and is frequently mutated in many different types of human cancers. The COOH terminus of p53 modulates the transcriptional and apoptotic activities of the protein. Although COOH-terminal mutants of p53 are uncommon, we proposed that these p53 mutants nevertheless contributed to the selective clonal expansion of the cancer cells. Therefore, we analyzed the tumor-derived p53 COOH-terminal domain (CTD) mutants (352D/H, 356G/W, 342-stop, 360-del, and 387-del) functionally. The results have revealed that all mutants have impaired apoptotic activity when compared with wild-type p53. However, some of these mutants still transcriptionally transactivate p21Waf/Cip1 and inhibit cell growth. Interestingly, of the tumor-derived CTD mutants, oligomerization-defective mutant 342-stop was the only one that did not exhibit sequence-specific DNA binding or failed to transactivate p21Waf1/Cip1, Bax, and IGF-BP3 transcriptionally. The failure to inhibit cell growth by this tumor-derived CTD mutant supports the hypothesis that p53 sequence-specific transcriptional transactivity to p21Waf1/Cip1 is correlated with induction of cell cycle arrest and that the p53 transcriptional transactivity requires oligomerization of the p53 protein. These and other data indicate that the CTD of p53 is an important component of p53-mediated apoptosis and cell growth arrest and that inactivation of the apoptotic function, but not the inhibition of growth, is an important step during human tumorigenesis.

Apoptosis↗
PubMed1998

Immunity to p53 induced by an idiotypic network of anti-p53 antibodies: generation of sequence-specific anti-DNA antibodies and protection from tumor metastasis.

The general overexpression of p53 by different types of tumor cells suggests that p53 immunity might be generally useful for tumor immunotherapy. We describe here the induction of immunity to p53 and resistance to tumor metastasis using an idiotypic network. Mice were immunized with domain-specific anti-p53 monoclonal antibodies (Ab1): PAb-248 directed to the N-terminus; PAb-246 directed to the specific DNA-binding region; or PAb-240 directed to a mutant p53 that does not bind specific DNA. Immunized mice responded by making anti-idiotypic antibodies (Ab2) specific for the Ab1 inducer. Ab1 PAb-246 induced Ab2 that, like p53 itself, could bind the specific DNA oligonucleotide sequence of the p53 responsive element. Mice immunized with Ab1 PAb-240 or PAb-246 spontaneously made Ab3 anti-p53 antibodies that reflected the specificity of their Ab1 inducers: Ab1 PAb-246 induced Ab3 specific for wild-type p53; PAb-240 induced Ab3 specific for mutant p53. Ab1 PAb-248 induced only Ab2. The spontaneously arising Ab3 were of T cell-dependent IgG isotypes. Peptides from the complementarity determining regions of the Ab1 antibodies PAb-240 and PAb-246 could also induce Ab3 anti-p53. Finally, mice that produced Ab3 anti-p53 acquired resistance to tumor metastases. Therefore, an anti-idiotypic network built around certain domains of p53 seems to be programmed within the immune system, specific Ab2 antibodies can mimic the DNA binding domain of p53, and Ab3 network immunity to p53 can be associated with resistance to tumor cells.

Amino Acid Sequence↗
PubMed1998

Mutant p53 protein expression interferes with p53-independent apoptotic pathways.

Loss of normal p53 function was found frequently to interfere with response of cancer cells to conventional anticancer therapies. Since more than half of all human cancers possess p53 mutations, we decided to explore the involvement of mutant p53 in drug induced apoptosis. To further evaluate the relationship between the p53-dependent and p53-independent apoptotic pathways, and to elucidate the function of mutant p53 in modulating these processes, we investigated the role of a p53 temperature-sensitive (ts) mutant in a number of apoptotic pathways induced by chemotherapeutic drugs that are currently used in cancer therapy. To that end, we studied the M1/2, myeloid p53 non-producer cells, and M1/2-derived temperature-sensitive mutant p53 expressing clones. Apoptosis caused by DNA damage induced with gamma-irradiation, doxorubicin or cisplatin, was enhanced in cells expressing wild type p53 as compared to that seen in parental p53 non-producer cells; mutant p53 expressing clones were found to be more resistant to apoptosis induced by these factors. Actinomycin D, a potent inhibitor of transcription, as well as a DNA damaging agent, abrogated the restraint apoptosis mediated by mutant p53. These observations suggest that while loss of wild type p53 function clearly reduces the rate of apoptosis, p53 mutations may result in a gain of function which significantly interferes with chemotherapy induced apoptosis. Therefore, to achieve a successful cancer therapy, it is critical to consider the specific relationship between a given mutation in p53 and the chemotherapy selected.

Animals↗
PubMed1998

Idiotypic immunization induces immunity to mutated p53 and tumor rejection.

The p53 molecule might serve as a common tumor-associated antigen, as the tumor suppressor gene p53 is mutated and the p53 protein is often over-expressed in tumor cells. We report that effective immunity to p53 can be induced through an idiotypic network by immunization of mice with a monoclonal antibody (PAb-240) specific for mutated p53, or with a peptide derived from the complementarity determining region (CDR) 3 of the variable domain of the light chain (VL) of this antibody. The immunized mice produced IgG antibodies to p53 and mounted a cytotoxic reaction to a tumor line bearing mutated p53. The idiotypically immunized mice were resistant to challenge with the tumor cells. Thus antibodies to p53 might serve as immunogens for activating resistance to some tumors. At the basic level, these findings indicate that a network of p53 immunity may be organized naturally within the immune system.

Animals↗
PubMed1998

DNA-binding activity of wild-type p53 protein is mediated by the central part of the molecule and controlled by its C terminus.

The DNA binding activity of wild type p53 is central to its activity. The "central" part of the molecule, where most mutations appear in primary human tumors, is the actual DNA binding domain. The C-terminal part was shown to exert a negative effect on the DNA binding activity. In the present study we show that while anti-p53 antibodies recognizing the C terminus of the wild type p53 facilitate DNA binding activity, blocking of the wild type specific epitope by specific anti-p53 antibodies, inhibited the DNA binding activity of the wild type p53 protein. An alternatively spliced p53 protein exhibits an augmented DNA binding activity. The fact that most p53 mutants have lost the wild type p53 conformation specific epitope, coupled with the observation that blocking of this site by binding specific antibodies, prevents the interaction of wild type p53 with DNA, suggests that maintaining the correct structural conformation of this site is central for DNA binding activity. Still, the internal structure of the p53 target and particularly the length of the sequence between the two tandem inverted repeats, is critical for protein-DNA interaction behavior.

Antibodies, Monoclonal↗
PubMed1997

PACT: cloning and characterization of a cellular p53 binding protein that interacts with Rb.

Cellular functions of tumor suppressor proteins can be mediated by protein-protein interactions. Using p53 as a probe to screen an expression library, a cDNA encoding a 250 kDa protein was isolated. Recombinant forms of this protein, designated PACT, bind to wild type p53 while two different mutations abolish this interaction. PACT protein can also interfere with p53 specific DNA binding. PACT contains a serine/arginine (SR) rich region and a C' terminal lysine rich domain. The 250 kDa PACT protein can be precipitated from cell lysates by a method specific for SR proteins. snRNPs can be co-immunoprecipitated from cells with anti-PACT antibodies. These antibodies stain cell nuclei in a speckled pattern reminiscent of the distribution of known splicing factors. Recently, RBQ1, a truncated human homologue of PACT was identified by virtue of Rb binding. We show that RBQ1 is truncated as a result of a possible mutational event. PACT can interact with both cellular Rb and p53.

Alternative Splicing↗
PubMed1997

The murine C'-terminally alternatively spliced form of p53 induces attenuated apoptosis in myeloid cells.

The onset of p53-dependent apoptosis results from the accumulation of damaged DNA. Recently, it was shown that the C' terminus of the p53 protein plays a central role in sensing damaged DNA. In our present study, we examined the role of the C' terminus in the induction of apoptosis. A temperature-sensitive (ts) mutant of the alternatively spliced form of p53 (p53AS-ts) and the ts mutant of the regularly spliced form (p53RS-ts) were used to generate series of stable clones with increasing amounts of p53 protein. Apoptotic patterns induced by either the regularly spliced p53 product (p53RS) or a C'-terminally alternatively spliced p53 product (p53AS) were compared. We found that although both forms of p53 induced apoptosis following expression of the wild-type protein conformation, the kinetics were different. Apoptosis induced by the p53AS protein was attenuated compared to that induced by p53RS. The delay in the manifestation of the apoptotic features following p53AS expression was in agreement with a delay in the regulation of the expression of apoptosis-related genes. The observation that p53 with an altered C' terminus is still capable of inducing apoptosis suggests that the actual onset of the apoptotic process most probably involves structural domains other than the C' terminus of the p53 molecule. However, the fact that the apoptotic activity mediated by the p53AS product was slower than that mediated by the p53RS product suggests that the C' terminus indeed exerts a certain control on the apoptotic activity of the p53 molecule.

Alternative Splicing↗
PubMed1997

The DNA binding regulatory domain of p53: see the C.

The p53 tumor suppressor gene is a critical regulator of normal development involved in cell cycle control pathways, such as growth arrest, differentiation and apoptosis. The DNA binding activity of p53 is central to its function. In addition to the specific DNA binding activity that is confined to the "core" domain of the molecule, the C-terminus seems to play an important role in both controlling the specific as well as exhibiting a non-specific DNA binding activity, which is directly associated with sensing damaged DNA. The C-terminal DNA binding activity appears to be regulated by phosphorylation, glycosylation, splicing and binding of several factors. The C-terminus seems to recognize single and double stranded DNA breaks that occur during DNA replication and recombination, as well as following external DNA stress signals. Unless the cell manages to correct the DNA damage it has the tempting option to progress towards apoptosis. Imagine the C-terminus as a traffic light ensuring the safe "on going" through the cell cycle; in case damaged DNA could not be corrected, p53 dependent apoptosis or terminal differentiation "signs" are turned on!

Animals↗
PubMed1996

The role of p53 in the induction of polyploidity of myelomonocytic leukemic M1/2 cells.

p53 was shown to play a central role in the maintenance of genomic integrity. The present experiments suggest that p53 is involved in the control of cell ploidity. Using a p53 non-producer cell line, M1/2, that was reconstituted to express either wild type or mutant p53 protein, by infection with the temperature sensitive (Ts) p53Val135 virus, it was found that both loss of wild type p53 or overexpression of mutant p53, may be associated with the generation of cell polyploidity. Overexpression of mutant p53 protein enhanced the appearance of giant cells that further accumulated following gamma-irradiation. Expression of wild type p53 reduced the level of giant cells which accumulated in the parental M1/2 p53 non-producer cells following gamma-irradiation. This activity of the wild type p53 seems to be mediated by either the reduction in the rate of giant cell generation, as observed in M1/2 derived cell lines expressing low levels of wild type p53 protein or by facilitating their apoptosis, as observed in wild type p53 high-producer cells. The latter conclusion is further supported by the observation that isolated giant cells are directly induced to undergo apoptosis following wild type p53 expression.

Animals↗
PubMed1995

Augmented DNA-binding activity of p53 protein encoded by a carboxyl-terminal alternatively spliced mRNA is blocked by p53 protein encoded by the regularly spliced form.

DNA-binding activity of the wild-type p53 is central to its function in vivo. However, recombinant or in vitro translated wild-type p53 proteins, unless modified, are poor DNA binders. The fact that the in vitro produced protein gains DNA-binding activity upon modification at the C terminus raises the possibility that similar mechanisms may exist in the cell. Data presented here show that a C-terminal alternatively spliced wild-type p53 (ASp53) mRNA expressed by bacteria or transcribed in vitro codes for a p53 protein that efficiently binds DNA. Our results support the conclusion that the augmented DNA binding activity of an ASp53 protein is probably due to attenuation of the negative effect residing at the C terminus of the wild-type p53 protein encoded by the regularly spliced mRNA (RSp53) rather than acquisition of additional functionality by the alternatively spliced C' terminus. In addition, we found that ASp53 forms a complex with the non-DNA-binding RSp53, which in turn blocks the DNA-binding activity of ASp53. Interaction between these two wild-type p53 proteins may underline a mechanism that controls the activity of the wild-type p53 protein in the cell.

Alternative Splicing↗
PubMed1995

The DNA binding activity of wild type p53 is modulated by blocking its various antigenic epitopes.

Interaction of wild type p53 with specific DNA target sequences, which is dictated by several structural domains, can be modified by blocking the different antigenic epitopes of the protein. Comparison of p53 protein expressed by recombinant bacteria (wtp53-Bac) to that produced in an eukaryotic system by a vaccinia expression vector (wtp53-Vac), indicated that only the later exhibited spontaneous DNA-binding activity. Furthermore, DNA-binding patterns of these wild type p53 proteins were affected differently by their interactions with monoclonal anti-p53 antibodies recognizing individual antigenic epitopes of the molecule. While the vaccinia derived p53 that spontaneously bound DNA is supershifted by the N'-terminal specific antibodies PAb-248, the bacterial derived p53 protein that retains this antigenic epitope but does not bind DNA spontaneously, is not affected. The C'-terminal specific PAb-421 antibodies accelerated binding of the bacterial p53 protein and modified the pattern of the interaction of the vaccinia derived p53 DNA. DNA-binding patterns generated by PAb-421 and PAb-248, suggest that either interaction of wild type p53 is dependent on modification of the p53 protein or that it interacts with cellular factors which their activity can be mimicked by PAb-421. Saturation of both types of wild type p53 with several anti-p53 monoclonal antibodies directed against the wild type p53 specific epitope that maps to the N'-terminal border of the DNA-binding region, blocked specific DNA-binding. The fact that most p53 mutants have lost the wild type p53 conformation specific epitope coupled with the observation that blocking of this site by binding specific antibodies, prevents the interaction of wild type p53 with DNA, suggests that maintaining the correct structural conformation of this site is central for DNA-binding activity. The wild type specific epitope which maps to the N'-terminal border of the DNA-binding region is neighboring the first beta-strand detected by the recent crystallographic analysis.

Animals↗
PubMed1994

Does wild-type p53 play a role in normal cell differentiation?

Inactivation of the p53 tumor suppressor gene plays a major role in malignant transformation. The central question in this issue is concerned with the understanding of the function of p53 in normal cells and its deregulation in cancer cells. Several in vitro and in vivo experimental models have indicated that induction of cells to undergo differentiation involve up-regulation in the expression of the p53. In the case of B cell differentiation, p53 was found to be involved in several steps of the differentiation pathway. The conclusion that p53 plays a role in normal development and differentiation in vivo is substantiated by the observation that p53 is expressed during embryonic development and is detected at low levels in a number of organs of adult mice. Accentuated levels of p53 in testes of adult mice, suggests that p53 plays a role in the meiotic process of spermatogenesis. B cell differentiation and spermatogenesis are biological pathways which normally involve DNA reshuffling and rearrangements. In accordance with the notion that p53 is associated with DNA repair it is tempting to speculate that at least in these physiological pathways p53 functions as a master gene that controls genome integrity.

Animals↗