Translocation t(14;18) and gain of chromosome 18/BCL2: effects on BCL2 expression and apoptosis in B-cell non-Hodgkin's lymphomas.

Gain of chromosome 18q and translocation t(14;18) are] frequently found in B-cell non-Hodgkin's lymphomas (B-NHL). Increased BCL2 transcription and BCL2 protein expression have been suggested to be the result of the gain. We utilized FISH, PCR and array CGH to study BCL2 and chromosome 18 copy number changes and rearrangements in 93 cases of B-NHL. BCL2 protein was expressed in >75% of the tumor cells in 92% of the cases by immunohistochemistry. Gain of BCL2 was associated with a 25% increase in BCL2 expression levels (immunoblotting), whereas t(14;18) resulted in a 55% increase in BCL2 levels compared to cases without BCL2 alterations. The tumor cell (spontaneous) apoptotic fractions were similar for the cases with different BCL2 genotypes. However, the normal cell apoptotic fractions were higher for the tumors with t(14;18) compared to the tumors without BCL2 alterations, while the tumors with gain of BCL2 only showed intermediate levels. Low-level gains of parts of chromosome 18 were found in 14 of the 38 B-NHL cases with t(14;18), with a consensus region 18pter-q21.33 that did not include the BCL2 gene. The 11 cases with 18q gain only showed a consensus region encompassing 18q21.2-18q21.32 and 18q21.33, which contain PMAIP1/MALT1 and BCL2, respectively.

Proliferation-dependent expression and phosphorylation of pRB in B cell non-Hodgkin's lymphomas: dependence on RB1 copy number.

Some studies have suggested that a significant fraction of non-Hodgkin's lymphomas (NHL) do not express pRB protein, possibly due to deletions of RB1. We examined RB1/centromere 17 copy number by fluorescent in situ hybridisation, and pRB expression/phosphorylation by immunohistochemistry (IHC) and immunoblotting (IB) in 66 cases of B cell NHL. Thirteen cases had lost one RB1 copy relative to centromere 17 copy number and total DNA content. Case 458/88 had no RB1 copies. pRB levels were heterogeneous as assessed by IB (0.04-1.12 relative units), but all tumours, except for case 458/88, expressed pRB localised to the nucleus in >75% of the tumour cells by IHC. The fraction of phosphorylated pRB was correlated with pRB expression (r(2)= 0.56, P < 0.001). The 14 cases with loss of RB1 had lower pRB expression (median 0.25) than those without (median 0.48, P < 0.001), but a correlation with S phase fraction (r(2) = 0.43, P < 0.001; previously published data for tumour-specific S phase and apoptotic fractions) indicated that the variation in pRB expression was due to differences in proliferative activity. Furthermore, the regression lines for pRB expression vs S phase fraction were not different for the cases with or without loss of one RB1 copy (P = 0.5). Cases 154/88 (one RB1 copy) and 258/88 (two RB1 copies), in addition to case 458/88, had low expression of (hypophosphorylated) pRB (0.04, 0.08 and 0.04), despite their high S phase fractions (21%, 17% and 21%). There was no association between pRB expression/RB1 copy number and apoptotic fraction. Neither pRB expression nor loss of RB1 had prognostic value, but cases 154/88, 258/88, and 458/88 had short survival times (5, 3 and 46 months, respectively) compared to the others (median survival: 44 months, P = 0.03). It is suggested that pRB expression and function are normal in 63 of 66 NHL cases, including 12 of 13 lymphomas with loss of one RB1 allele.

Loss of chromosome 11q21-23.1 and 17p and gain of chromosome 6p are independent prognostic indicators in B-cell non-Hodgkin's lymphoma.

Comparative genomic hybridization (CGH) was employed to study chromosomal aberrations in relation to cell proliferation, apoptosis, and patient survival in 94 cases of B-cell non-Hodgkin's lymphoma diagnosed between 1983 and 1993. Eighty cases had aberrations by CGH. Chromosomal regions 1p21-31.1 (10%), 6cen-q24 (12%), 8p (11%), 9p21-ter (14%), 11q21-23.1 (11%), 13q13-21.1 (12%), and 17p (15%) were frequently lost. Gains were found at 3q21-ter (22%), 6p (11%), 7p (12%), 8q23-ter (13%), 12cen-q15 (17%), 17q24-ter (13%), and 18q13.3-21 (20%). A high number of aberrations (> or = 4, 33 cases) was associated (P < or = 0.001) with the mantle cell and diffuse large B-cell lymphoma subtypes, a high fraction of tumour cells in S phase, and short survival (RR (relative risk) = 3.7). Loss of 1p21-31.1, 8p, 9p21-ter, 11q21-23.1, and 13q13-21.1 were associated with mantle cell lymphoma (P < or = 0.03), while gain of 6p and 12cen-q15 were more frequent in diffuse large B-cell and small lymphocytic lymphoma, respectively (P = 0.04). Loss of 8p and 17p, and gain of 3q21-ter, 6p, 7p, and 8q23-ter were associated with a high S phase fraction (P < or = 0.03), but none of the aberrations were associated with tumour apoptotic fraction (P > or = 0.13). The most important prognostic CGH parameters (P < 0.001) were losses of 11q21-23.1 (RR = 3.8) and 17p (RR = 4.4), and gain of 6p (RR = 4.2). The latter parameters and IPI were the only ones with independent prognostic value (RR = 10, 5.0, 6.7, and 3.7, respectively; P < 0.001) when assessed together with lymphoma sub-type, primary versus relapse cases, treatment, B symptoms, S phase fraction, and presence of BCL1 and BCL2 translocations. A combined CGH/IPI binary parameter had high prognostic value for patients receiving different treatments, with various lymphoma sub-types, and for primary as well as relapse cases.

Oncogenic aberrations in the p53 pathway are associated with a high S phase fraction and poor patient survival in B-cell Non-Hodgkin's lymphoma.

The implications of aberrations in the p53 pathway for induction of apoptosis and regulation of S phase entry, and for patient survival, were investigated in 83 B-cell Non-Hodgkin's lymphomas. Eight cases had missense mutations in exons 5, 7, 8 and 9 as revealed by constant denaturant gel electrophoresis and sequencing. Fifteen cases had lost 1 TP53 allele as revealed by fluorescent in situ hybridization and comparative genomic hybridization. Ten cases expressed high levels of p53 as assessed by immunoblotting and immunohistochemistry. S phase fractions were higher, apoptotic fractions were the same and survival times were shorter in all aberration groups compared with the cases with no TP53/p53 aberrations. Since many tumors had more than one TP53/p53 aberration, the tumors were divided into groups with the following characteristics: no TP53/p53 aberrations; loss of one TP53 allele only (9 cases), TP53 point mutation (8 cases), high-level p53 expression and no TP53 mutation (3 cases). Tumors from the 3 latter groups had higher median S phase fractions (5%, 7.6%, and 5%, respectively, p<0.02) than the cases without any aberrations (1.1%), and survival time for these patients was much shorter (relative risks of 5.9, 8.9, and 6.6, respectively, p<0.003). Apoptotic fractions were similar in all these groups (p=0.09). Multivariate analysis showed that the presence of TP53/p53 aberrations is a strong and independent prognostic parameter in B-cell Non-Hodgkin's lymphoma.

Proliferation and apoptosis in malignant and normal cells in B-cell non-Hodgkin's lymphomas.

We have examined apoptosis and proliferation in lymph node cell suspensions from patients with B-cell non-Hodgkin's lymphoma using flow cytometry. A method was developed which allowed estimation of the fractions of apoptotic cells and cells in the S-phase of the cell cycle simultaneously with tumour-characteristic light chain expression. Analysis of the tumour S-phase fraction and the tumour apoptotic fraction in lymph node cell suspensions from 95 B-cell non-Hodgkin's lymphoma (NHL) patients revealed a non-normal distribution for both parameters. The median fraction of apoptotic tumour cells was 1.1% (25 percentiles 0.5%, 2.7%). In the same samples, the median fraction of apoptotic normal cells was higher than for the tumour cells (1.9%; 25 percentiles 0.7%, 4.0%; P = 0.03). The median fraction of tumour cells in S-phase was 1.4% (25 percentiles 0.8%, 4.8%), the median fraction of normal cells in S-phase was significantly lower than for the tumour cells (1.0%; 25 percentiles 0.6%, 1.9%; P = 0.004). When the number of cases was plotted against the logarithm of the S-phase fraction of the tumour cells, a distribution with two Gaussian peaks was needed to fit the data. One peak was centred around an S-phase fraction of 0.9%; the other was centred around 7%. These peaks were separated by a valley at approximately 3%, indicating that the S-phase fraction in NHL can be classified as 'low' (< 3%) or 'high' (> 3%), independent of the median S-phase fraction. The apoptotic fractions were log-normally distributed. The median apoptotic fraction was higher (1.5%) in the 'high' S-phase group than in the 'low' S-phase group (0.8%; P = 0.02). However, there was no significant correlation between the two parameters (P > 0.05).

Hypoxia-induced apoptosis in human cells with normal p53 status and function, without any alteration in the nuclear protein level.

We have studied hypoxia-induced inactivation of cells from three established human cell lines with different p53 status. Hypoxia was found to induce apoptosis in cells expressing wild-type p53 (MCF-7 cells), but not in cells where p53 is either mutated (T-47D cells), or abrogated by expression of the HPV18 E6 oncoprotein (NHIK 3025 cells). Apoptosis was demonstrated by DNA fragmentation, using agarose gel electrophoresis of DNA and DNA nick end labeling (TUNEL). We demonstrate that extremely hypoxic conditions (< 4 ppm O2) do not cause any change of expression in the p53 protein level in these three cell lines. In addition, the localization of p53 in MCF-7 cells was found exclusively in the nucleus in only some of the cells both under aerobic and hypoxic conditions. Furthermore, no correlation was found between the p53-expression level and whether or not a cell underwent apoptosis. Flow cytometric TUNEL analysis of MCF-7 cells revealed that initiation of apoptosis occurred in all phases of the cell cycle, although predominantly for cells in S phase. Apoptosis was observed only during a limited time window (i.e., approximately 10 to approximately 24 h) after the onset of extreme hypoxia. While 66% of the MCF-7 cells lost their ability to form visible colonies following 15 h exposure to extreme hypoxia, only approximately 28% were induced to apoptosis, suggesting that approximately 38% were inactivated by other death processes. Commitment to apoptotic cell death was observed in MCF-7 cells even for oxygen concentrations as high as 5000 ppm. Our present results indicate that the p53 status in these three tumor cell lines does not have any major influence on cell's survival following exposure to extremely hypoxic conditions, whereas following moderate hypoxia, cells expressing functional p53 enhanced their susceptibility to cell death. Taken together, although these results suggest that functional p53 might play a role in the induction of apoptosis during hypoxia, other factors seem to be equally important.

Uncoupling of the order of the S and M phases: effects of staurosporine on human cell cycle kinases.

The protein kinase inhibitor staurosporine (SSP) was employed to study the involvement of kinases in human cell cycle progression. Thirty to 100 ng/ml SSP blocks entry into S phase and M phase. Lack of entry into S phase is due to impaired activity of the retinoblastoma protein kinase. The requirement for any of the SSP-sensitive kinases for cell cycle progression can be abrogated in tumour cells. Therefore, these kinases act in a checkpoint network negatively controlling the initiation of S phase, M phase and cytokinesis, rather than being inherent parts of a substrate-product chain required for the initiation of the cell cycle phases. As a consequence of the lack of certain checkpoint effectors, tumour cells may endoreduplicate or binucleate in the presence of SSP. The latter processes, as well as meiosis, are naturally occurring in specialized cell types, leading to the idea that this checkpoint network controls the order of the cell cycle phases in normal cells. A model is presented where the cell cycle is envisioned as two independently running cycles, the S and the M cycle, which are controlled by intra and intercycledependent checkpoints in human somatic cells. The model accounts for the dependency of S and M phase initiation on the successful completion of the previous M and S phase, respectively, as well as entry into a resting state.

A comparison of different modes for the detection of p53 protein accumulation. A study of bladder cancer.

The aim of the present study was to evaluate different techniques for the analysis of p53 protein accumulation in human bladder cancer. The accumulation was evaluated in 23 carcinomas by immunoblotting (IB), immunohistochemistry (IHC) and flow cytometry (FCM). The results revealed that six (26%), eight (35%) and ten (43%) of the tumours were p53 protein positive by IB, IHC and FCM, respectively. Mutation analysis of the TP53 gene confirmed mutations in 8 of 9 tumours which showed increased levels of p53 protein by FCM. Our results indicate that IHC could be applied for studies of p53 protein accumulation in archival formalin fixed, paraffin-embedded bladder tumours. However, FCM is a more sensitive and objective method for the detection of p53 protein than IHC and this should be taken into account when routinely evaluating the p53 protein accumulation by IHC.

P-glycoprotein is not expressed in a majority of colorectal carcinomas and is not regulated by mutant p53 in vivo.

Overexpression of the MDR1 product, P-glycoprotein (Pgp), has been shown to be one of the mechanisms underlying the development of multidrug resistance (MDR). Recently, one mutant p53 has been shown to stimulate the MDR1 gene promoter in vitro, whereas wild-type p53 repressed this activity. We measured Pgp and p53 expression by immunoblotting in 34 colorectal tumours, and performed mutation analyses on the p53-positive tumours to confirm the presence of mutant p53 protein. Tumour DNA indices (DIs) were also measured using flow cytometry. Pgp was detected in 44% (15/34) of the tumours and in 100% (13/13) of the normal mucosas (P = 0.0005), with highest levels of expression seen in normal mucosa, suggesting that initial drug resistance in colorectal tumours is not caused by Pgp. Highly DNA aneuploid tumours demonstrated the lowest levels of Pgp expression relative to moderately aneuploid and diploid colorectal tumours. p53 protein was detected in 53% (18/34) of the tumours, and 12 of 14 p53-positive tumours had p53 gene mutations, p53-negative tumours had approximately twice the level of Pgp expression of p53-positive tumours. Pgp expression was not associated with either p53 expression (P = 0.73) or incidence of p53 gene mutation (P = 0.70), suggesting that mutant p53 does not induce Pgp overexpression in colorectal carcinomas.

The retinoblastoma gene product is bound in the nucleus in early G1 phase.

The product of the retinoblastoma susceptibility gene (pRB) exerts its growth-regulatory effects during the G1 phase of the cell cycle, where all pRB present has been assumed to be in the underphosphorylated form. We demonstrate here that pRB is underphosphorylated and firmly bound in the nucleus only in early G1 phase. All G0 cells contain bound, underphosphorylated pRB. The duration of the cell cycle and of the G1 phase seems to be determined by the time during which pRB is underphosphorylated and bound in the nucleus. The observed time lag between the phosphorylation and release of pRB in the G1 phase and entry into S phase was 6.5 h and independent of the G1 transit time. The data suggest that pRB is not directly involved in initiation of DNA replication.

P53 expression is associated with a high-degree of tumor DNA aneuploidy and incidence of p53 gene mutation, and is localized to the aneuploid component in colorectal carcinomas.

p53 protein expression was studied by immunoblotting in 34 colorectal carcinomas and 28 of the corresponding normal mucosas, and correlated with tumor DNA ploidy as measured by flow cytometry. p53 protein was detected in 35% (12/34) of the tumors; the normal mucosas were negative. Fifty-five percent (12/22) of the tumors examined for mutations within the four hotspots (exons 5-8) of the p53 gene had point mutations. p53 expression correlated significantly with the presence of p53 gene mutations; 67% (8/12) of the tumors with mutations expressed p53, whereas only one of 10 tumors where no mutations were detected expressed the protein (p=0.01). Four tumors with p53 gene mutations did not express p53. Fifty-nine percent (20/34) of the tumors were aneuploid. p53 expression correlated significantly with aneuploidy; a total of 55% (11/20) of the aneuploid tumors were positive for p53 compared to 7% (1/14) of the diploid tumors (p=0.009). All of the 11 highly aneuploid tumors (1.31 less-than-or-equal-to DNA index (DI); less-than-or-equal-to 1.86) expressed p53, whereas all of the 9 moderately aneuploid tumors (1.11 less-than-or-equal-to DI less-than-or-equal-to 1.29) were p53-negative. Flow cytometry was also used to resolve cell cycle- and ploidy specific p53 expression in nuclei in 4 aneuploid tumors. p53 expression in these tumors was confined to the aneuploid component, whereas the diploid component was negative. p53 was seen in nuclei in all phases of the cell cycle of proliferating aneuploid cells. Neither p53 expression nor tumor DNA ploidy were correlated with Dukes' stage (p=1.00 and 0.72, respectively). The data suggest that high levels of mutant p53 may play a causative role in the generation of highly aneuploid tumors.

Photodynamic effects and hyperthermia in vitro.

Cells from the established human line NHIK 3025 were labelled with hematoporphyrin derivative in vitro. Subsequently, the cells were treated with light and hyperthermia. The cells could be irradiated either before, during or after the incubation at a hyperthermic temperature. It was shown that hyperthermia given shortly after the light exposure gave a synergistic killing effect. In spite of some loss of porphyrins from the cells, the light sensitivity increased 20 min after a light irradiation. At later times, the cells apparently repaired some of the photodynamic damage at 37 degrees C. At higher temperatures, the repair was inhibited.

Effects of haematoporphyrin derivative and light in combination with hyperthermia on cells in culture.

Interactions between the photodynamic effect of haematoporphyrin derivative and hyperthermia are reported. Cells labelled with haematoporphyrin derivative and irradiated with red light were sensitized by heat, particularly when the cells were heated after the exposure to light. It is shown that there is a synergistic interaction between the photodynamic effect and hyperthermia (42.5 and 45 degrees C). Hyperthermia-induced inhibition of the repair of photodynamic damage is suggested as a mechanism for the interaction. The possibility that these findings may be advantageous to cancer therapy is discussed.

Photodynamic effects on human cells exposed to light in the presence of hematoporphyrin. pH effects.

Human cells derived from a carcinoma in situ (NHIK 3025) were exposed in vitro to visible light and hematoporphyrin at different pH levels. The cells were inactivated more efficiently at pH 6.7 and 7.2 than at pH 7.8 The treatment with light and hematoporphyrin also induced DNA damage more efficiently at the former pH values than at the latter one. The variation in the efficiency of the photodynamic effect with the pH is mainly due to the fact that the cellular uptake of hematoporphyrin increases with decreasing pH.