Search PubMed⌕ Search

Biomedical subjects

K G Wiman

Publications and source records attributed to K G Wiman.

At least 55 records · Page 3Linked to original sources
PubMed1995

Resting B-cells, EBV-infected B-blasts and established lymphoblastoid cell lines differ in their Rb, p53 and EBNA-5 expression patterns.

Using immunofluorescence technique we have analysed the Rb, p53, EBNA-2 and EBNA-5 expression pattern in EBV infected human B-cells and established lymphoblastoid cell lines (LCL-s). Resting B-cells showed only a faint Rb and no p53 immunostaining. The expression of both Rb and p53 increased after EBV infection. The change was first detectable 6 h after infection. The frequency of brilliantly Rb positive cells increased more rapidly than p53 positives. EBNA-2 and EBNA-5 became first detectable 12 h after infection. The frequency of EBNA positive cells in the freshly infected cultures was concordant with the proportion of CD23 and PCNA positives, but remained consistently below the frequency of Rb and p53 positive cells. Double immunofluorescence staining showed that all EBNA-5 positive cells were strongly Rb and p53 positive. LCL-s did not stain for p53, whereas the Rb staining was maintained at a high level. The EBNA-5 staining pattern changed from brilliant almost homogeneous nuclear staining in the freshly infected B-cells, to a nonhomogeneous pattern with a small number of strongly fluorescent nuclear bodies in established LCL-s. There was no change in the EBNA-2 staining pattern. Our findings indicate that the immortalization of B-cells by EBV may initially involve a high expression of EBNA-5, p53 and Rb, but only cells with low p53 and focal expression of EBNA-5 in nuclear bodies have the selective advantage required to grow into immortalized lines.

Antigens, Viral↗
PubMed1995

p53 binds single-stranded DNA ends through the C-terminal domain and internal DNA segments via the middle domain.

We have previously reported that wild-type p53 can bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules. Here we demonstrate that p53 can also bind to internal segments of ss DNA molecules via a binding site (internal DNA site) distinct from the binding site for DNA ends (DNA end site). Using p53 deletion mutants, the internal DNA site was mapped to the central region (residues 99-307), while the DNA end site was mapped to the C-terminal domain (residues 320-393) of the p53 protein. The internal DNA site can be activated by the binding of ss DNA ends to the DNA end site. The C-terminal domain alone was sufficient to catalyze DNA renaturation, although the central domain was also involved in promotion of renaturation by the full-length protein. Our results suggest that the interaction of the C-terminal tail of p53 with ss DNA ends generated by DNA damage in vivo may lead to activation of non-specific ss DNA binding by the central domain of p53.

DNA, Single-Stranded↗
PubMed1995

What is the restriction point?

The restriction point (R) separates two functionally different parts of G1 in continuously cycling cells. G1-pm represents the postmitotic interval of G1 that lasts from mitosis to R. G1-ps represents the pre S phase interval of G1 that lasts from R to S. G1-pm is remarkably constant in length (its duration is about three hours) in the different cell types studied so far. G1-ps, however, varies considerably, indicating that entry into S is not directly followed after passage through R. Progression through G1-pm requires continuous stimulation by mitogenic signals (e.g. growth factors) and a high rate of protein synthesis. Interruption of the mitogenic signals or moderate inhibition of protein synthesis leads to a rapid exit from the cell cycle to G0 in normal (untransformed) cells. Upon restimulation with mitogenic signals, the cell returns to the same point in G1-pm from which it left the cell cycle. Thus the cell seems to have a memory for how far it has advanced through G1-pm, suggesting that a continuous structural alteration, for example chromatin decondensation, takes place in G1. The molecular background to transition from growth factor dependence in G1-pm to growth factor independence in G1-ps (a switch which represents commitment to a new cell cycle and passage through R) is still not fully understood. Cyclin-dependent kinase (cdk)-mediated hyperphosphorylation of the retinoblastoma protein (Rb), and concomitant liberation (and activation) of members of the E2F family of transcription factors, are probably important aspects of R control in normal cells. A key component here could be cdk2 activity which is controlled by cyclin E. When cdk2 activity starts to increase rapidly in G1, due to activation of a positive feedback loop, it reaches a critical level above which cdk inhibitors (CKIs) such as p21 and p27 are outweighed; the cell has then become independent of mitogenic and inhibitory signals and is committed to a new cell cycle. However, other components are probably also involved in R control. For instance, a 'cryptic' R (a G1-pm-like state) can be induced even in tumour cells that do not respond to growth factor starvation or protein synthesis inhibitors, and are therefore probably defective in the cdk-Rb-E2F pathway. Possibly, a certain degree of chromatin decondensation has to take place after mitosis in order to allow transcription of, for example, the cyclin E gene or other critical E2F targets. Although the molecular basis for restriction point control still remains unclear, we can expect rapid progress in this important field over the next few years.

Cell Cycle↗
PubMed1995

Infrequent MDM2 gene amplification and absence of gross WAF1 gene alterations in nasopharyngeal carcinoma.

We have investigated the possible involvement of MDM2 and WAF1 gene alterations in the development of nasopharyngeal carcinoma (NPC). MDM2 and WAF1 were analysed in 46 primary NPCs by Southern blot analysis. Forty-five tumours showed a normal EcoRI hybridisation pattern and hybridisation intensity with a human MDM2 cDNA probe. One tumour had more intense normal size MDM2 hybridising bands. Densitometric scanning revealed a 10-12-fold MDM2 gene amplification, as compared with human placenta DNA. All 46 tumours showed normal size WAF1 EcoRI bands that hybridised with normal intensity. This is the first demonstration of MDM2 gene amplification in NPC. Nonetheless, our analysis indicates that gross structural alterations of the MDM2 and WAF1 genes are infrequent events in the genesis of NPC.

Blotting, Southern↗
PubMed1995

Characterization of human buccal epithelial cells transfected with the simian virus 40 T-antigen gene.

Serum-free cultures of normal human buccal epithelial cells were transfected with a plasmid containing the SV40 T-antigen (SV40T) gene. Two major lines developed that showed extended lifespans (between 30 and 40 weeks) as compared with the controls (approximately 6 weeks). Continued growth through one or two crises generated several sublines. They expressed the epithelial marker keratin and also exhibited nuclear expression of SV40T. The lines showed abnormal karyotypes with both numerical and structural aberrations and variably responded to agents that normally inhibit growth and/or induce terminal differentiation, i.e. transforming growth factor-beta 1 and fetal bovine serum. One of the lines, termed SVpgC2a, developed into an apparently immortal line, since it had undergone more than 700 population doublings from over 2 years in culture. Further characterization of this line demonstrated its clonal origin, with integration of two copies of SV40T at the same site and the presence of both normal retinoblastoma and wild-type p53 proteins. This line showed high resistance to growth inhibition by transforming growth factor-beta 1 and serum similar to that shown by buccal carcinoma cell line SqCC/Y1. Neither SVpgC2a nor its parental lines were tumorigenic when injected into athymic nude mice, whereas the SqCC/Y1 cells induced tumors. The various lines with extended but finite lifespans, complemented by one immortalized line, which retained non-malignant properties upon extended culture, provide a battery of model systems that will be useful for studying mechanisms of human oral carcinogenesis.

Animals↗
PubMed1995

Reversible nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 and hsp70 proteins after exposure to heat shock or cell density congestion.

The Epstein-Barr virus (EBV)-encoded, nuclear matrix-associated EBNA-5 protein is preferentially localized within distinct nuclear blobs in EBV-immortalized lymphoblastoid cell lines. We have previously found that the same blobs also contain retinoblastoma (Rb) protein. We now show that they contain hsp70 protein as well. Both EBNA-5 and hsp70 translocate to the nucleolus under cell density congestion or after heat shock. Both proteins relocate to their original position upon the re-establishment of normal physiological conditions. EBNA-5 is tightly bound to the nuclear matrix. The translocated EBNA-5 is also tightly associated with matrix structures, as shown by sequential elution-based cell fractionation. The Rb protein does not translocate to the nucleolus. The virally encoded EBNA-1, -2, -3 and -6, and cellular PCNA, snRNP and cyclin E are not affected either. The translocation of EBNA-5 to the nucleolus is not species- or cell type-specific since stress conditions induced the same phenomenon in EBNA-5-transfected human, mouse and rat cells of different tissue origins.

Animals↗
PubMed1995

bcl-2 inhibits wild-type p53-triggered apoptosis but not G1 cell cycle arrest and transactivation of WAF1 and bax.

We have earlier shown that wild-type (wt) p53 expressed from a temperature-sensitive construct (ts p53) triggers apoptosis in the v-myc retrovirus-induced, p53-negative T-cell lymphoma line J3D (Y. Wang et al., Cell Growth & Differ., 4: 467-473, 1993). We also found that constitutive bcl-2 expression inhibits wt p53-triggered apoptosis in these cells (Y. Wang et al., Oncogene, 8: 3427-3431, 1993). Here we demonstrate that more than 90% of the ts p53-transfected J3D cells were arrested in G1 at 18 h after induction of wt p53 expression by temperature shift to 32 degrees C. At this time, at least 80% of the cells remained viable. After 30 h at 32 degrees C, around 50% of the cells had died by apoptosis, while most of the remaining cells were still alive in G1, indicating that p53-induced apoptosis occurred following G1 arrest. The G1 cell cycle arrest at 18 h after temperature shift to 32 degrees C was reversible, as shown by the fact that the cells readily resumed exponential growth following temperature shift back to 37 degrees C, although viability dropped from around 80 to 65%. Expression of both WAF1 and bax mRNA was induced by wt p53 in both the ts p53 and ts p53/bcl-2 transfected cells. The kinetics of G1 cell cycle arrest at 32 degrees C was similar in both the ts p53 and the ts p53/bcl-2 double transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Progressive B-cell chronic lymphocytic leukaemia frequently exhibits aberrant p53 expression.

We have analysed the p53 status in non-progressive and progressive chronic B-cell leukemia (B-CLL) by ELISA, immunoprecipitation, FACS and cDNA sequencing in relation to in vitro proliferation in response to Staphylococcus aureus strain Cowan I (SAC) and IL-2. In FACS, cells from progressive leukaemia were found to over-express p53 with a conformation recognized by PAB240. In a PAb240-based ELISA, 60% of progressive B-CLL were positive. DNA sequencing of p53 exons 5 to 9 revealed a codon 179 His to Gln change in one of the ELISA-positive, progressive B-CLL but failed to reveal any mutations in 4 other ELISA-positive, progressive B-CLL. Among progressive B-CLL populations, 10/14 responded by proliferation in vitro to SAC/IL-2. In non-progressive cells, low levels of p53 were found by FACS, none was positive in the PAb240 ELISA and only one case showed a weak proliferative response to SAC/IL-2. Low p53 expression was also seen in different types of normal B cells, resting and activated, and in EBV-transformed B-cell lines, in contrast to the high expression observed in Burkitt lymphoma cell lines with verified p53 mutations. We conclude that progressive B-CLL is characterized by aberrant p53 expression which may be a significant prognostic factor.

Adult↗
PubMed1994

p53 binds single-stranded DNA ends and catalyzes DNA renaturation and strand transfer.

The p53 tumor-suppressor protein has previously been shown to bind double-stranded and single-stranded DNA. We report that the p53 protein can bind single-stranded DNA ends and catalyze DNA renaturation and DNA strand transfer. Both a bacterially expressed wild-type p53 protein and a glutathione S-transferase-wild-type p53 fusion protein catalyzed renaturation of different short (25- to 76-nt) complementary single-stranded DNA fragments and promoted strand transfer between short (36-bp) duplex DNA and complementary single-stranded DNA. Mutant p53 fusion proteins carrying amino acid substitutions Glu-213, Ile-237, or Tyr-238, derived from mutant p53 genes of Burkitt lymphomas, failed to catalyze these reactions. Wild-type p53 had significantly higher binding affinity for short (36- to 76-nt) than for longer (> or = 462-nt) single-stranded DNA fragments in an electrophoretic mobility-shift assay. Moreover, electron microscopy showed that p53 preferentially binds single-stranded DNA ends. Binding of DNA ends to p53 oligomers may allow alignment of complementary strands. These findings suggest that p53 may play a direct role in the repair of DNA breaks, including the joining of complementary single-stranded DNA ends.

Base Sequence↗
PubMed1994

Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts.

The expression of the retinoblastoma susceptibility (RB-1) gene was investigated in highly proliferating mouse embryonic stem (ES) cells and in slowly proliferating mouse embryonic fibroblasts. The RB protein was expressed at the same level in these two cell types. Mainly hyperphosphorylated RB was detected in exponentially-growing ES cells. Embryonic fibroblasts and embryonic stem cells were synchronized by colcemid block followed by mitotic shake-off. In embryonic fibroblasts, DNA replication started 10-15 h after exit from mitosis and RB was transiently dephosphorylated during the G1 phase as previously described. In ES cells, DNA replication started 2 h after release from the colcemid block but virtually no hypophosphorylated RB was observed after the release. Instead, there was a dramatic decrease in the total RB protein level between exit from mitosis and entry into S phase. These observations were made by using two different monoclonal antibodies, both in immunoblotting and immunoprecipitation experiments. Absence of hypophosphorylated RB and cell cycle-dependent change in total RB protein level may be relevant to the high proliferation rate and to the tumorigenic nature of mouse embryonic stem cells.

Animals↗
PubMed1993

EBNA-5, an Epstein-Barr virus-encoded nuclear antigen, binds to the retinoblastoma and p53 proteins.

Epstein-Barr virus (EBV) immortalized human lymphoblastoid cell lines express six virally encoded nuclear proteins, designated EBV nuclear antigens 1-6 (EBNA-1-6). We show that the EBNA-5 protein (alternatively designated EBNA-LP) that is required for B-cell transformation can form a molecular complex with the retinoblastoma (RB) and p53 tumor suppressor proteins. Using EBNA-5 deletion mutants, we have found that a 66-amino acid-long peptide, encoded by the W repeat of the EBV genome, is sufficient for binding. Point mutations of RB and p53 that inhibit their complexing with other DNA viral oncoproteins do not affect their binding to EBNA-5. p53 competes with RB for EBNA-5 binding. Our data suggest that the mechanisms involved in EBV transformation may include impairment of RB and p53 function.

Amino Acid Sequence↗
PubMed1993

Position-dependent nuclear accumulation of the retinoblastoma (RB) protein during in vitro myogenesis.

The expression of the retinoblastoma (RB) protein has been studied during in vitro muscle differentiation by immunofluorescence staining with three different antibodies against RB protein. Proliferating mononucleate L6 rat myoblasts showed a low level of expression. As cells began to enter a nonreplicating G0 state, the cell population became heterogeneous. Some nonreplicating cells showed a high level of expression. Nuclei at the two ends of myotubes were strongly positive, whereas centrally located nuclei showed low RB expression. Overexpression of the human RB protein in rat L6 myotubes from a Semliki forest virus (SFV)-based, transient expression vector produced a similar picture. Terminally located nuclei expressed human RB at a much higher level than did the centrally located nuclei. The results suggest that individual nuclei with a multinucleated syncytium may undergo position-dependent specialization.

Animals↗
PubMed1993

The retinoblastoma gene: role in cell cycle control and cell differentiation.

The retinoblastoma (RB) gene is the prototype tumor suppressor gene. It encodes a nuclear protein that acts as a cell cycle control checkpoint at the G1 phase. Deletion or inactivation of both RB alleles plays an essential, rate-limiting role in retinoblastoma and in the osteosarcomas that arise within families that carry a mutated RB gene. RB inactivation is also found in other sarcomas, small cell carcinoma of the lung, and in carcinoma of the breast, bladder, and prostate. Transforming proteins encoded by SV40, and the transforming or tumor-associated subtypes of adenoviruses and human papilloma viruses (HPV) can bind RB, thereby blocking its normal function. The EBNA-5 protein of Epstein-Barr virus (EBV) is also able to bind RB in vitro. In addition, RB can interact with several cellular proteins, including the transcription factor E2F. RB gene knock-out mice die in utero around day 14 of gestation. The embryos show disturbed neural and hematopoietic differentiation, indicating that RB is vitally important for these processes. This notion is further supported by studies demonstrating that RB expression in mouse embryo tissues is highest in cells undergoing differentiation, and that RB is required for MyoD-induced muscle differentiation.

Animals↗
PubMed1993

Wild-type p53-triggered apoptosis is inhibited by bcl-2 in a v-myc-induced T-cell lymphoma line.

Using a temperature sensitive p53 construct (ts p53), we have earlier shown that expression of wild-type (wt) p53 triggers apoptosis in a v-myc-induced T-cell lymphoma line that lacks endogenous p53, and in a Burkitt lymphoma line that carries mutant p53. We have suggested that apoptosis is elicited by the contradictory signals emanating from the constitutively activated myc gene and the growth arresting signal of wt p53 (Ramqvist et al., 1993; Wang et al., 1993). Work in other laboratories has shown that constitutive c-myc expression can induce apoptosis when cell proliferation is inhibited due to the lack of growth stimulating factors. Expression of bcl-2 could inhibit apoptosis. In order to test whether p53-induced apoptosis can be prevented by bcl-2, we have introduced a retrovirally driven bcl-2 construct into our v-myc-induced murine T-cell lymphoma line, previously transfected with ts p53. About 90% of the parental ts p53 transfected cells died of apoptosis within 3 days after induction of wt p53 expression at 32 degrees C. Two clones of ts p53/bcl-2 double transfectants that expressed high levels of bcl-2 from the introduced construct were completely protected from apoptosis, following transfer of the cells to 32 degrees C. One clone that expressed the exogenous bcl-2 only at a low level was partially protected from wt p53-induced apoptosis. Clones of the parental ts p53 carrying cells transfected with the puromycin resistance gene vector, without the bcl-2 gene underwent 90% apoptosis. These results suggest that bcl-2 may prevent apoptosis in cells simultaneously exposed to the proliferation-stimulating effect of activated myc and the growth arresting signal of wt p53.

Animals↗
PubMed1993

Reconstitution of wild-type p53 expression triggers apoptosis in a p53-negative v-myc retrovirus-induced T-cell lymphoma line.

Inactivation or mutation of the p53 tumor suppressor gene has been observed in a wide variety of human and murine tumors. We have found that a v-myc retrovirus (J3)-induced T-cell lymphoma line (J3D) has lost one of its p53 alleles, whereas the other has become inactivated due to the insertion of a Moloney murine leukemia provirus in intron 4 with an opposite transcriptional orientation. No p53 protein could be detected by immunoprecipitation with monoclonal anti-p53 antibodies. We have transfected this line with the temperature-sensitive murine Val135 construct that is expressed as mutant p53 at 37 degrees C and largely wild-type p53 at 32 degrees C. There was no difference in the number of viable cells among the p53 transfectants, the parental cells, and neomycin vector-transfected control cells at 37 degrees C. Following a temperature shift to 32 degrees C, the p53 transfectants rapidly lost viability, and 95-100% of the cells were dead by 3 days, whereas the control cells remained unaffected. Examination of DNA isolated from p53-transfected cells grown at 32 degrees C revealed nucleosomal fragmentation, indicating cell death by apoptosis. It is suggested that apoptosis is triggered by contradictory signaling. Constitutively expressed v-myc can stimulate cell proliferation, whereas expression of wild-type p53 in cells that have lost endogenous p53 expression in the course of their neoplastic development may suppress growth.

Alleles↗
PubMed1993

Wild-type p53 induces apoptosis in a Burkitt lymphoma (BL) line that carries mutant p53.

All Burkitt lymphoma (BL) biopsies and cell lines carry a c-myc/Ig translocation. The resulting constitutive activation of c-myc is regarded as an essential factor for the progressive growth of the tumor cells. At least 60% of BL cell lines carry a mutated p53 gene as well. It has been shown that the growth of mutant p53 carrying tumor cells could be inhibited by the introduction of wild-type p53. In order to examine whether this also applies to the presumably 'myc-driven' BL cell, we have transfected the Epstein-Barr virus (EBV) negative BL41 cell line with a temperature sensitive p53 mutant (p53-Val135) that expresses p53 with a largely mutant conformation at 37.5 degrees C and mostly wild-type conformation at 32 degrees C. At 37.5 degrees C, the p53-Val135 transfected cells behaved like the parental or neo transfected control cells. However, expression of exogenous wild-type p53 at 32 degrees C resulted in a rapid reduction of the number of viable cells while the parental and neo control cells remained unaffected. Cell death was due to apoptosis as shown by chromatin and nuclear condensation and specific DNA fragmentation. The first signs of apoptosis were evident after 10 h at 32 degrees C and after 3 days 90-100% of the cells had undergone apoptosis. These findings indicate an incompatibility between expression of wild-type p53 and progressive growth of BL cells if their neoplastic development has included a p53 mutation. The question whether apoptosis was induced in by the wild-type protein per se or by the contradictory signals of a constitutively activated c-myc and wild-type p53 needs further investigation.

Apoptosis↗
PubMed1992

[Tumor suppressor genes: mutations in RB and p53 genes are significant carcinogenic factors].

The RB and p53 tumour suppressor genes encode nuclear proteins that exert an inhibitive effect on cell growth. A large variety of human tumour types manifest loss or mutation of the RB or p53 genes, and p53 mutation is the commonest genetic alteration found in tumour cells. In addition, the RB and p53 proteins may be inactivated by complex formation with viral oncoproteins--for instance, in the case of cervical carcinoma carrying human papilloma virus. In vivo introduction of an intact RB or p53 gene into malignant cells lacking the respective gene results in suppression of the neoplastic phenotype and thus of tumourigenicity, p53 being the more potent of the two in this respect. Further elucidation of tumour suppressor genes may well result in future improvement in the diagnosis and treatment of cancer.

Genes, Retinoblastoma↗