Topoisomerase II (topo II) is the target enzyme of etoposide, and DNA–topo II complex accumulation is considered crucial for the cytotoxic effect. We used a SDS–KCl precipitation assay to determine the complex accumulation induced by etoposide in leukaemic cells isolated from 58 patients, 31 with acute myelogenous leukaemia (AML), and 27 with chronic lymphocytic leukaemia (CLL). To investigate whether the sensitivity towards etoposide was dependent on the complex accumulation in the cells, we investigated the drug-induced DNA damage using a DNA unwinding assay and the in vitro cytotoxicity of etoposide using the MTT assay. AML cells had higher complex accumulation (P=0.006) and more DNA damage (P=0.029) compared with CLL cells. The data support a relationship between etoposide-induced complex accumulation and DNA damage in leukaemic cells from AML and CLL patients. However, the induced DNA damage did not translate to in vitro cytotoxicity, suggesting that other factors, such as DNA repair and apoptosis functions, also play important roles to determine the etoposide sensitivity.

Cross‐resistance between different classes of anti‐neoplastic agents can jeopardize successful combination cancer chemotherapy. In this study, we observed an unexpected cross‐resistance between the podophyllotoxine derivative etoposide (VP) and the nucleoside analogue cladribine (CdA) in CCRF‐CEM cells developed for resistance to VP. The resistant cells also displayed 14‐ and twofold resistance to cytarabine (ara‐C) and gemcitabine respectively. Closer analysis of these cells showed that they contained lower amounts of topoisomerase (topo) IIα ( P < 0·001) and β protein ( P < 0·026), formed substantially lower amounts of the topo II–DNA complex, and had a markedly decreased level of Fas (CD95/APO‐1)‐ligand mRNA expression. Interestingly, Fas expression in the resistant cells did not differ from that in the parental cell line. No differences were observed in the accumulation/efflux of daunorubicin or in the gene expressions of P‐glycoprotein, multidrug resistance‐associated protein and the lung resistance‐related protein. The activity of deoxycytidine kinase (dCK), responsible for activation of CdA and ara‐C, was the same for resistant and wild‐type cells. However, there was an increase in the activity of the cytosolic 5′‐nucleotidases (5′‐NT), responsible for deactivation of nucleotides, amounting to 206% ( P < 0·001) for the high K m and 134% ( P < 0·331) for the low K m 5′‐NT in resistant cells. The high K m 5′‐NT is probably responsible for the decreased amount of the active metabolite CdA 5′‐triphosphate [40% decreased ( P < 0·045)], as well as for other purine ribonucleosides and deoxyribonucleosides triphosphates in the resistant cells. In contrast, a significantly higher deoxycytidine triphosphate (dCTP) level (167%, P < 0·001) was observed in the resistant cells. Thus, this study suggests that the major cause of resistance to the nucleoside analogues CdA and ara‐C in cells selected for resistance to VP is a result of metabolic alterations producing increased activity of 5′‐NT and higher dCTP levels. Furthermore, these results indicate that there is a common factor in the regulation of nucleotide‐degrading enzymes and DNA topoisomerases, which may be altered in cross‐resistant cells.

Elevated expression of the membrane transporter p‐glycoprotein (pgp) and impaired expression of the nuclear enzyme topoisomerase II (topo II) are well‐known mechanisms for in vitro acquired drug resistance. The clinical relevance of topo II remains unclear, whereas a relationship between pgp levels and treatment results has been shown in acute myelogenous leukaemia (AML). We have investigated the relationships between the levels of topo II and pgp, and in vitro sensitivity to etoposide in mononuclear blood cells from 24 patients with AML, 16 with chronic lymphocytic leukaemia (CLL) and five healthy blood donors.Following incubation with etoposide, AML cells showed more DNA damage, determined by a DNA unwinding technique, than CLL cells (P = 0.001), whereas there was no difference in cellular etoposide accumulation. Pgp and topo IIβ levels, determined by Western blot, showed a pronounced variation between patients, but no correlation with induced DNA damage, whereas topo IIα protein was undetectable. In the AML group, topo IIβ expression correlated with pgp expression (ρ = 0.7, P = 0.001, n = 24). The topo IIβ expression was 147.4(±74.6)% in the pgp+ AML cells (n = 10), compared to 33.4(±27.8)% in pgp− AML cells (n = 14) (P = 0.0001). Our results show a previously unknown coexpression of topo IIβ and pgp in AML, thereby suggesting that topo IIβ is a potentially interesting resistance factor in AML.

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.

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)

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.

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.

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.

From a lymphoid tumour induced by 7,12-dimethylbenz-[a]-anthracene (DMBA) + methyl-N-nitrose-N-urea (MNU) in an [AKR Rb(6.15) x CBAT6T6]F1 mouse, a macrophage- monocyte line (KT-10) was isolated. Following ethyl methanesulfonate (EMS) treatment, a bromodeoxyuridine (BUdR) resistant subline was selected. Serial propagation of this line in vitro in the presence of BUdR (28 months) with periodic cytogenetic and molecular examinations, has led to the definition of four successive stages. During stage I, the cells were trisomic for chromosome 15. They contained Rb(6.15) and Rb(del6.15) of AKR and T(14;15) of CBA origin. Southern blotting showed the presence of both germline (G) and rearranged (R) c-myc. At stage II, Rb(del6.15) has duplicated. Both Rb(6.15) and T(14;15) persisted together with G-myc and R-myc. In stage III, the CBA-derived T(14;15) was lost, in parallel with G-myc. At this stage, a Dic.In(6.15) was detected. One of its arms was cytogenetically identical with the long arm of In(6.15) in the variant IgK/myc translocations. This chromosome carried R-myc and IgK in juxtaposition, as indicated by comigration on pulsed field electrophoresis (PFGE). At stage IV, the R-myc carrying AKR-derived chromosome 15s were present in six copies. Possible relationships between the increasing R/G myc ratio and changed growth characteristics in vivo and in vitro are discussed.

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.

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.

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.

Mouse plasmacytomas induced by pristane oil alone, or in combination with Abelson murine leukemia virus (A-MuLV), regularly carry one of three alternative chromosomal translocations that juxtapose c-myc to immunoglobulin heavy- or light-chain loci. E mu-c-myc transgenic mice develop translocation-free plasmacytomas after induction by pristane oil and/or A-MuLV [Sugiyama, H., Silva, S., Wang, Y., Weber, G., Babonits, M., Rosen, A., Wiener, F. & Klein, G. (1990). Int. J. Cancer, 46, 845-852]. In order to test whether another member of the myc family, N-myc, could play a similar role as c-myc, we treated E mu-N-myc transgenic mice with pristane and helper-free A-MuLV. Of 20 mice that received a single pristane injection followed by A-MuLV, 17 developed plasmacytomas with a mean latency period of 54 +/- 20 days. In a corresponding group that only received a single pristane injection, five out of six transgenic mice developed plasmacytomas with a mean latency period of 142 +/- 32 days. However, after three monthly injections of pristane, all 15 transgenic mice developed plasmacytomas with a mean latency period of 128 +/- 20 days. All plasmacytomas expressed the N-myc transgene, while none of them expressed either c-myc or endogenous N-myc. None of the tumors carried the usual plasmacytoma-associated translocations.