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K G Wiman

Publications and source records attributed to K G Wiman.

At least 37 records · Page 2Linked to original sources
PubMed1998

New p53-based anti-cancer therapeutic strategies.

The p53 gene is frequently mutated in human tumours and therefore an important target for therapeutic intervention. Several p53-based strategies for treatment of cancer are currently under development. p53 gene therapy has resulted in tumour regression in patients with lung cancer. A mutant adenovirus can obliterate tumour cells carrying mutant p53 or lacking p53, but is unable to replicate in normal cells. Furthermore, current studies suggest that reactivation of mutant p53 proteins in tumours using small p53-activating molecules may initiate p53-dependent apoptosis and thus eliminate the tumour.

Adenoviridae↗
PubMed1997

Wig-1, a new p53-induced gene encoding a zinc finger protein.

Wild type p53 expressed from a temperature-sensitive (ts p53) construct induces both G1 cell cycle arrest and apoptosis in the p53-negative J3D mouse T lymphoma line (Wang et al., 1995). Using differential display analysis, we have identified one new p53-induced gene, wig-1 (for wild type p53-induced gene 1), whose 7.6 kb and 2.2 kb transcripts are upregulated in ts p53-transfected J3D cells following induction of wild type p53 expression by temperature shift to 32 degrees C. The wig-1 transcripts were also induced in irradiated NIH3T3 and p21-/- fibroblasts but not in irradiated p53-/- fibroblasts. Whole body gamma irradiation caused induction of both wig-1 transcripts in mouse brain, testis, kidney, spleen and lung. A basal wig-1 expression was detected in brain, testis and kidney. The WIG-1 protein contains three zinc finger motifs and a putative nuclear localization signal.

Amino Acid Sequence↗
PubMed1997

[New transcription factor-based cancer therapy. Gene therapy, cell lysis, reactivation of p53 are the new approaches].

The p53 protein is a transcription factor that triggers cell cycle arrest and cell death (apoptosis) in response to certain types of cellular stress, including DNA damage. Human tumours carry p53 mutation in about 50 per cent of cases. Rapid advances in recent years have opened up the possibility of new p53-based approaches to cancer therapy. In a gene therapy approach, the insertion of an intact p53 gene in tumour cells carrying mutant p53 has been shown to result in tumour regression in lung cancer patients. A mutant adenovirus has been shown to kill tumour cells carrying mutant p53 or lacking p53, but not to affect normal cells. Moreover, it may be possible to restore the normal function of mutant p53 proteins by using small p53-activating molecules and thus inducing p53-dependent apoptosis in tumour cells.

Apoptosis↗
PubMed1997

Restoration of the growth suppression function of mutant p53 by a synthetic peptide derived from the p53 C-terminal domain.

We demonstrate here that synthetic 22-mer peptide 46, corresponding to the carboxy-terminal amino acid residues 361-382 of p53, can activate specific DNA binding of wild-type p53 in vitro and can restore the transcriptional transactivating function of at least some mutant p53 proteins in living cells. Introduction of peptide 46 in Saos-2 cells carrying a Tet-regulatable His-273 mutant p53 construct caused growth inhibition and apoptosis in the presence of mutant p53 but not in its absence, confirming that the effect of the peptide is mediated by reactivation of mutant p53. Moreover, peptide 46 caused apoptosis in mutant as well as wild-type p53-carrying human tumor cell lines of different origin, whereas p53 null tumor cells were not affected. These findings raise possibilities for developing drugs that restore the tumor suppressor function of mutant p53 proteins, thus selectively eliminating tumor cells.

Apoptosis↗
PubMed1997

Role of p53 mutation in polyomavirus-induced tumorigenesis.

Small DNA tumour viruses, such as simian virus 40 (SV40), papilloma viruses and adenoviruses, encode proteins that form complexes with and inactivate the p53 and retinoblastoma (RB) proteins. This convergent evolution reflects the common need of these viruses to inactivate these two important regulators of cell cycle progression and cell survival. Polyomavirus, a close relative of SV40, is different. Its large T protein complexes only with RB, not with p53. We have examined whether this is compensated by the frequent appearance of p53 mutations in polyomavirus-induced tumours. We tested the p53 status of 15 polyomavirus-induced sarcomas. Two sarcomas were p53-negative while six carried mutant p53. Another six sarcomas expressed low levels of wild-type p53. One tumour expressed high levels of wild-type p53 protein as shown by DNA sequencing and immunofluorescence staining. MDM2 amplification was not detected in any of the tumours, but Northern blotting showed that MDM2 was overexpressed in at least two tumours that expressed wild-type p53 and in one tumour that expressed both wild-type and mutant p53. Treatment with the DNA-damaging agent mitomycin C caused p53 protein accumulation followed by induction of MDM2 and WAF1/p21 mRNA in four of the tumours expressing wild-type p53, indicating that p53-mediated transcriptional activation was unaltered in these tumours. However, p53-mediated transactivation of WAF1/p21 was impaired in the wild-type p53-expressing tumours that expressed elevated levels of MDM2. These results demonstrate that p53 mutation and inactivation are frequently but not invariably involved in polyomavirus-induced tumorigenesis.

Animals↗
PubMed1997

Decreased immunoglobulin deposition in tumors and increased immature B cells in p53-null mice.

Recent studies have hinted that there may be a relationship between p53 and the immune response. In preliminary experiments, we found significantly decreased levels of immunoglobulin deposition in 13 of 16 p53-null tumors compared with 2 of 17 tumors derived from p53 +/- mice. We further explored the effect of p53 on B-cell development and function. p53-null mice contained more splenic white pulp and more immature B cells in the bone marrow compared with p53 +/- mice. p53-null B cells were hyperresponsive to proliferative challenge but were not more resistant to signal-induced apoptosis. Several p53 DNA-binding sites were localized to the regulatory regions of immunoglobulin heavy and light chain genes, including the KII site, which serves as an enhancer for rearrangement of the mouse kappa chain J cluster genes. Levels of p53 protein and the kappa chain sterile transcript increased after exposure of pre-B cells to the DNA damaging agents etoposide and Adriamycin. Our observations suggest that p53 may be involved in B-cell maturation and may relay certain stress signals to affect B-cell function.

Animals↗
PubMed1996

The single-stranded DNA end binding site of p53 coincides with the C-terminal regulatory region.

p53 is a transcription factor that binds double-stranded (ds) DNA in a sequence-specific manner. In addition, p53 can bind the ends of single-stranded (ss) DNA. We previously demonstrated that ssDNA oligonucleotides interact with the C-terminal domain of p53 and stimulate binding to internal segments of long ssDNA by the p53 core domain. Here we show that the p53 C-terminal domain can recognize staggered ss ends of dsDNA. We have mapped the binding site for ssDNA ends to residues 361-382 in human p53 using a p53 deletion mutant (p53-delta 30) lacking the 30 C-terminal amino acid residues and a series of 22mer peptides. The binding site for DNA ends coincides with a region previously implicated in regulation of sequence-specific DNA binding by the core domain. The interaction of the C-terminal regulatory domain with the ends of ssDNA or with the protruding ends of dsDNA stimulates both sequence-specific and non-specific DNA binding via the core domain. Electron microscopy demonstrated the simultaneous binding of p53 to dsDNA and a ssDNA end. These results suggest a model in which interaction of the p53 C-terminal tail with DNA ends generated after DNA damage causes activation of sequence-specific p53 DNA binding in vivo and may thus provide a molecular link between DNA damage and p53-mediated growth arrest and apoptosis.

Amino Acid Sequence↗
PubMed1996

Wild-type p53-induced apoptosis in a Burkitt lymphoma cell line is inhibited by interferon gamma.

The tumor suppressor p53 plays a central role in negative growth control, including growth arrest and apoptosis. Interferons (IFNs) are capable of modulating a variety of cellular responses, including apoptosis. In this study, we have evaluated the influence of gamma- and alpha-interferon (IFN) on wild-type (wt) p53-induced apoptosis using a Burkitt lymphoma cell line, BL41, transfected with a temperature-sensitive p53 construct, gamma-IFN, but not alpha-IFN, was found to protect cells from wt p53-induced apoptosis. The gamma-IFN-dependent protection was due neither to down-regulation of p53, nor to the p53-induced genes, p21 (WAF-1) and bax, nor to up-regulation of bcl-2 or bcl-xL. Expression of the proto-oncogene c-myc, implicated in the control of both proliferation and apoptosis, was not affected by gamma-IFN. We conclude that gamma-IFN can suppress p53-induced apoptosis, and that the cytokine microenvironment may be decisive in the cellular response to wt p53 expression.

Apoptosis↗
PubMed1996

Abrogation of p53-induced G1 arrest by the HPV 16 E7 protein does not inhibit p53-induced apoptosis.

Wild type (wt) p53 expressed from a temperature-sensitive construct (ts p53) can induce both G1 cell cycle arrest and apoptosis in the p53-negative J3D mouse T lymphoma line (Wang et al, 1995). The human papillomavirus (HPV) 16 E7 protein has been shown to prevent p53-induced G1 cell cycle arrest following DNA damage. We asked whether inhibition of p53-induced G1 arrest by overexpression of the HPV16 E7 protein in the ts p53-transfected J3D cells would interfere with p53-induced apoptosis. Whereas a majority of the ts p53-expressing J3D cells were arrested in the G1 phase 22 h after induction of wt p53 by temperature shift to 32 degrees C, the E7/ts p53-expressing cells showed only a minor increase in the number of cells in G1 at this time point. In addition, the E7/ts p53-expressing cells showed a much less dramatic reduction in number of cells in S phase than the ts p53-expressing cells. This demonstrates that E7 at least partially rescues the cells from p53-induced G1 arrest. In contrast, overexpression of HPV16 E7 did not have any effect on the kinetics nor the frequency of p53-triggered apoptotic death, as shown by FACS analysis, trypan blue exclusion, and DNA fragmentation analysis. These findings support the notion that p53-induced G1 arrest and p53-induced apoptosis are two separate independent pathways.

Animals↗
PubMed1996

Frequent expression of Bcl-2 in renal-cell carcinomas carrying wild-type p53.

The p53 tumor-suppressor gene is the most commonly mutated gene in cancer. However, p53 gene alterations are infrequent in renal-cell cancer (RCC). Bcl-2 has been shown to inhibit apoptosis triggered by wild-type p53 and an inverse correlation between Bcl-2 expression and p53 mutation has been observed in breast cancer and glioma. To characterize the expression of bcl-2 in RCC and its relationship to the p53 status, we analyzed 25 RCCs by immunohistochemistry for Bcl-2 and p53, Southern hybridization for bcl-2, and PCR-SSCP and sequencing for p53. Positive Bcl-2 staining was detected in 17 of 25 RCCs, whereas positive p53 staining was seen in only 1. Amplification of bcl-2 or p53 mutation was not detected in any of the tumors. Bcl-2 protein was expressed in all 7 RCC cell lines examined. Only one of the 7 lines had p53 mutation. These results suggest that overexpression of bcl-2, rather than p53 mutation, may prevent apoptosis during RCC development.

Apoptosis↗
PubMed1996

Cyclin E overexpression in relapsed adult acute lymphoblastic leukemias of B-cell lineage.

Using Western blot analysis, we examined cyclin E and cyclin A protein levels in 19 patients with acute lymphoblastic leukemia ([ALL] 15 B-ALL and four T-ALL). Whereas normal, nonproliferating peripheral blood mononuclear cells (PBMCs) expressed low levels of the 50-kD cyclin E, ALL blasts in the peripheral blood, although showing low-level or no proliferation as judged by FACS/cell-cycle analysis and cyclin A protein levels, expressed high levels of cyclin E, with a mean value similar to that of the proliferating Burkitt's lymphoma cell line, Akata. The accumulation of a protein shown to shorten the G1 phase of the cell cycle, cyclin E, in growth-delayed leukemic blasts may reflect the malignant status of these cells. Before treatment, B-ALL cells expressed predominantly the 50-kD cyclin E. T-ALL samples displayed the 50-kD cyclin E protein and a smaller, approximately 43-kD cyclin E species. In paired B-ALL samples taken before treatment and at relapse, we found a significant overexpression of the 50-kD protein in relapsed samples (P < .006), plus the presence of up to four additional smaller-molecular-weight species of cyclin E, illustrating clear diagnosis versus relapse differences. Cyclin E expression in ALL blasts may correlate to the relative malignant status of the cells, with higher protein levels reflecting a more advanced stage of the disease and a greater potential to proliferate under permissive conditions.

Adolescent↗
PubMed1996

Phenotype-related differences in the expression of D-type cyclins in human B cell-derived lines.

Exposure of normal resting B lymphocytes to EBV in vitro leads to activation, subsequent immortalization, and the establishment of lymphoblastoid cell lines (LCLs). The endemic form of Burkitt's lymphoma (BL) is associated with EBV. EBV-positive BL lines maintain the original tumor phenotype (group I BL) initially and express only one EBV-encoded protein, EBV nuclear antigen (EBNA)-1. Most of them drift toward a LCL-like phenotype during in vitro culturing and express all nine EBV-encoded growth transformation-associated proteins (group III BL). Cyclin D2 and D3 have been found previously to differ in their mRNA expression in BL and LCL. Cyclin D2 expression has been attributed to EBV gene expression and to EBNA-2 and EBNA-5 in particular. We have studied cyclin D2/D3 expression in larger series of LCLs, BL lines, and freshly EBV-infected peripheral blood B lymphocytes, both at the mRNA and protein levels. The predominant cyclin D2 expression in the LCLs and group III BLs correlated with an activated B-cell phenotype. In contrast, only cyclin D3 was expressed in the group I BL lines. EBV-carrying group II BL lines that retained the BL-associated CD10 did not express cyclin D2. A collection of in vitro EBV-converted sublines of originally EBV-negative BLs that expressed a full set of the growth transformation-associated EBV proteins maintained their CD10 and cyclin D3 expression. Blood B lymphocytes expressed no D2 or D3 as judged by immunostaining. Cyclin D2 could be detected by immunostaining in a proportion of the activated blasts 40 h postinfection. Cyclin D3 that is expressed at a low level in the established LCLs was stained easily in B blasts at this time. The level of cyclin D3 at 72 h postinfection was two to three times higher than in the exponentially growing LCLs, as detected by immunoblotting. It was still 5-10 times lower than in group I BLs. Mitogen stimulation of primary B cells induced cyclin D3 at a similar level as in the LCLs. Our data are consistent with the notion of cell lineage-specific D-type cyclin expression. They also indicate that B cells may switch their D-type cyclin expression during differentiation.

B-Lymphocytes↗
PubMed1995

Mouse and rat B-myc share amino acid sequence homology with the c-myc transcriptional activator domain and contain a B-myc specific carboxy terminal region.

B-myc is a member of the myc gene family. Previous studies indicate that the rat B-myc gene contains a single exon which shows 77% nucleotide homology to the second exon of the rat c-myc gene. Its open reading frame (ORF) encodes a polypeptide with a predicted molecular weight of 20 kD. We have isolated a new, larger rat B-myc genomic clone. Sequence analysis of this clone confirmed the presence of one single coding exon. Furthermore, a genomic mouse B-myc clone was identified and compared to the rat homolog. Nucleotide analysis of B-myc coding and non-coding sequences suggests that it may be a functional gene evolved by selective duplication of part of the second c-myc exon. Analysis of the rodent B-myc open reading frames revealed two in-frame amino acid duplications in mouse B-myc and a 96% conservation at the amino acid level. Both rat and mouse B-myc proteins contain an identical and unique stretch of 14 carboxy terminal amino acid residues not found in other myc proteins. In vitro translation of rat and mouse B-myc ORF's yielded proteins that migrated as 26 kD bands in SDS-PAGE and could be immunoprecipitated by a polyclonal panmyc antiserum. Immunostaining of human lymphoma cells transiently transfected with a B-myc expression vector showed that the protein was mainly localized to the nucleus.

Amino Acid Sequence↗
PubMed1995

The EBV-encoded LMP1 protein inhibits p53-triggered apoptosis but not growth arrest.

We have previously shown that exogenous wild type p53 induces apoptosis in the Burkitt lymphoma line BL41 that carries endogenous mutant p53, using a temperature sensitive p53 construct expressed as mutant p53 at 37 degrees C and wild type p53 at 32 degrees C (Ramqvist et al., Oncogene, 8, 1495-1500, 1993). We also found that wild type p53-induced apoptosis is blocked by bcl-2 in a mouse T lymphoma line (Wang et al., Oncogene, 8, 3427-3431, 1993) The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) can protect Burkitt lymphoma cells from apoptosis induced by low serum. In order to test if LMP1 can block p53-triggered apoptosis, we infected BL41 cells expressing the ts p53 construct with an LMP1-carrying retrovirus. The LMP1-expressing BL41-ts p53 cells were arrested in G1 upon induction of wild type p53 expression at 32 degrees C, but did not enter apoptosis as shown by the absence of positive TUNEL staining. WAF1/p21 mRNA was induced at 32 degrees C in both the ts p53-expressing and ts p53/LMP1-expressing BL41 cells. Thus, LMP1 prevents p53-induced apoptosis but does not interfere with induction of WAF1/p21. The LMP1-infected cells expressed elevated bcl-2 protein levels. Therefore, our data suggest that LMP1 blocks p53-triggered apoptosis but not G1 arrest by upregulating bcl-2 expression.

Animals↗
PubMed1995

A splice donor site mutation results in the insertion of five extra amino acids into P53 from SEWA mouse sarcoma cells.

The status of the p53 gene in SEWA-AS12-ADH (S-ADH) cells, a subline of the mouse sarcoma cell line SEWA, was examined. Immunoprecipitation with wild-type (wt) or mutant P53-specific monoclonal antibodies (mAb) showed that both wt and mutant P53 were produced. Sequence analysis of the p53 cDNA and genomic DNA revealed a single nucleotide (nt) substitution at a splice donor site at the beginning of intron 7. As a result of this mutation, an alternative splice site 15 nt further 3' in intron 7 is used. The P53 protein translated from this aberrantly spliced mRNA carries an Arg258-->Ser substitution, followed by an insertion of 5 extra amino acids. This is the first example of a splice-site mutation in the mouse p53 gene.

Alternative Splicing↗
PubMed1995

RB-reconstituted human retinoblastoma cells form RB-positive intraocular and intracerebral but not subcutaneous tumors in SCID mice.

WERI-Rb27 human retinoblastoma cells were reconstituted with an intact RB gene by retrovirus-mediated gene transfer, in order to study the phenotypic effects of the protein in vitro and in vivo. Extensive morphological changes were observed, dominated by the formation of multinucleated giant cells. Six weeks after retroviral infection, the giant cells began to die and small cells emerged, resembling the parental non-reconstituted line. They expressed RB and continued to grow, although they showed an increased sensitivity to serum starvation. The original RB-negative cells grew progressively after subcutaneous inoculation into SCID mice, whereas the reconstituted cells failed to grow. RB-positive cells grew progressively in the corpus vitreum of the eye and in the brain, however. The RB-reconstituted cells grew more slowly and were less invasive than the parental cells and cells infected with a firefly luciferase (LUX) gene carrying retrovirus, used as controls. RB-reconstituted cells re-explanted from the intraocular and intracranial tumors continued to express full-length RB protein. RBeye2, an RB-positive cell line established from an eye tumor, was still unable to grow subcutaneously. The reduced tumorigenicity of the RB-reconstituted cells in the subcutaneous space may be due to the influence of locally acting growth-controlling signals or the absence of microenvironment-specific trophic factors. Alternatively, it may reflect the action of residual immune effectors in the SCID mice. If this is the case, these would have to be more effective at the subcutaneous site than in the eye or brain.

Animals↗