Expression and stabilization of the MCT-1 protein by DNA damaging agents

Abstract

The contribution of oncogene amplification and/or overexpression to T-cell lymphoid neoplasms has only of late been established with the implication of the TCL1 and MTCP1 genes in T-cell malignancies. Our laboratory has recently discovered a novel oncogene, MCT-1, amplified in a T-cell lymphoma and mapped to chromosome Xq22-24. MCT-1 has been shown to decrease cell-doubling time, dramatically shortening the duration of G₁ transit time and/or G1-S transition, and transforms NIH3T3 fibroblasts. Constitutive expression of MCT-1 results in a strong proliferative signal and is associated with deregulation of protein kinase-mediated G1/S phase checkpoints. In this study we analysed the level and subcellular localization of this novel cell cycle regulatory molecule as a function of cell cycle phase. In human lymphoid tumors expression of MCT-1 is constant throughout the cell cycle and remains cytoplasmic. Cells overexpressing MCT-1 have increased expression of cyclin D1 with dysregulation of the G₁-S checkpoint. Both cyclin D1 and MCT-1 are involved in regulating passage of cells through the G1 phase of the cell cycle. Since prior work has shown that gamma irradiation induces cyclin D1 expression we investigated the induction of MCT-1 to DNA damaging agents. We demonstrate that increases in MCT-1 protein in irradiated human lymphoid cells do not occur at the mRNA level and do not require new protein synthesis.

Introduction

Gene amplification is often associated with oncogenesis and drug resistance (Sandberg and Berger, 1994). The contribution of oncogene amplification and/or overexpression to T-cell lymphoid neoplasms has only of late been established (Ben-Yehuda et al., 1994). Recent reports have implicated the TCL1 and MTCP1 genes in leukemogenesis (Virgilio et al., 1994; Pekarsky et al., 1999; Sugimoto et al., 1999; Stern et al., 1993) Our laboratory has recently discovered a novel oncogene, MCT-1, amplified in a T-cell lymphoma and mapped to chromosome Xq22-24 (Prosniak et al., 1998). This region has been demonstrated to contain amplified DNA in a variety of lymphoid neoplasms using comparative genomic hybridization (CGH) (Monni et al., 1996; Werner et al., 1997). A recent report using both CGH and arbitrarily primed-PCR (AP–PCR) has identified several primary lymphomas with amplification of a common locus on chromosome X encompassing the region containing MCT-1 (Scarpa et al., 1999). MCT-1 has been shown to decrease cell doubling time, dramatically shortening the duration of G₁ transit time and/or G1-S transition, and transforms NIH3T3 fibroblasts (Prosniak et al., 1998; Dierov et al., 1999). While, there was no significant homology to MCT-1 at the primary sequence level using the BLAST program (Altschul and Lipman, 1990) there were interesting alignments at the structural protein level (Prosniak et al., 1998). The amino terminal half revealed a sequence identity of 32% with a domain of cyclin H that appears to specify protein-protein complexes (Andersen et al., 1997). An intriguing finding is that all IL-2 independent T-cell leukemia/lymphoma lines examined to date exhibited elevated MCT-1 protein levels in contrast to the IL-2 dependent T-cell lines which demonstrated low to absent MCT-1 protein levels (Dierov et al., 1999). The G₁/S mitogen IL-2 appears to push T-lymphocytes through G₁/S transition without requiring high levels of MCT-1 protein. This important association of growth factor independence with elevated MCT-1 protein levels in lymphoid tumor lines led us to examine the effect of MCT-1 overexpression on G₁ cyclin/cdk activity in MCT-1 transformed cell lines. We observed an increase in the kinase activity of both cdk4 and cdk6 in exponentially growing MCT-1 transformed cells compared to the parent control cells. An elevated level of cyclin D1 protein and increased G1 cyclin/cdk complex formation (Dierov et al., 1999) accompanied this increased kinase activity. These results demonstrated that constitutive expression of MCT-1 results in a strong proliferative signal and is associated with deregulation of protein kinase-mediated G1/S phase checkpoints.

Because the expression of cell cycle regulatory molecules is often regulated during cell cycle progression, the expression of MCT-1 may also be dependent on the phase of the cell cycle. In addition, the subcellular localization of some cell growth regulatory molecules has been shown to change depending on the phase of the cell cycle (Ye et al., 1998; Lam et al., 1997). In this study we analysed the level of MCT-1 protein as a function of cell cycle phase. We also examined the subcellular localization of MCT-1 in human lymphoid tumor cell lines using both asynchronous and synchronized cell populations.

We have previously demonstrated that cells overexpressing MCT-1 have increased expression of cyclin D1 with dysregulation of the G₁-S checkpoint (Dierov et al., 1999). This G₁-S checkpoint dysregulation is associated with cells progressing through the cell cycle despite sustaining DNA damage (reviewed in Paulovich et al., 1997). An earlier report has demonstrated that irradiation induces cyclin D1 protein levels and that those cells overexpressing cyclin D1 have an increased percentage in S phase compared to the parent cell line (Epperly et al., 1995). The strong proliferative signal provided by MCT-1 raised the possibility that it too might override the G₁/S checkpoint as well. To investigate the potential role of MCT-1 in this pathway we examined the induction of MCT-1 after exposure to DNA damaging agents.

Results

MCT-1 is predominantly localized to the cytoplasm in lymphoid cells and protein levels of MCT-1 are apparently stable throughout the cell cycle

We have examined the subcellular localization of MCT-1 in two human T-cell lymphoma cell lines using differential centrifugation followed by Western blotting with MCT-1 antibody of the various subcellular fractions. As demonstrated in Figure 1, MCT-1 was predominantly detected in the cytosolic fraction with slight staining in the heavy membrane fraction. We used the following antibodies; c-Myc, Bcl-2, NF-κB and Fas as controls for nuclear, heavy membrane, cytosolic and light membrane fractions, respectively.

Figure 1

Subcellular localization of MCT-1. Subcellular fractionation was performed with isolation of the following fractions: WC-whole cell extract, Nuc-nuclear, HM-heavy membranes enriched in mitochondria, LM-light membranes enriched in endoplasmic reticulum and S-100- soluble fraction. Immunobloting was carried out with the indicated primary antibodies. Observe the similar distribution of MCT-1 in both cell lines. Controls included: c-Myc, Bcl-2, NF-κB and Fas as controls for nuclear, heavy membrane, cytosolic and light membrane fractions, respectively

Some cell growth regulatory proteins show different levels of expression as the cell proceeds through the cell cycle. We examined the level of MCT-1 protein expression in lymphoid cell lines at different phases of the cell cycle. After arrest at the G1/S interphase using Aphidicolin, cells were released back into the cell cycle by washing and resuspension in growth medium. Levels of MCT-1 protein expression and cell cycle profile were assessed by Western blot and flow cytometry of propidium iodide-stained cells, respectively. MCT-1 protein levels in whole cell lysates essentially remained constant throughout cell cycle progression as demonstrated in Figure 2a. Similar results were obtained in two normal donor lymphocyte samples (data not shown).

Figure 2

Levels of MCT-1 protein and its subcellular localization remains constant throughout the cell cycle. (a) Jurkat cells are blocked at G₁/S phase boundary using 5 μg/ml Aphidicolin for 18 h. Following thorough washing cells were resuspended in complete RPMI and harvested at the indicated time points. Immunoblotting was performed with the indicated primary antibodies. Top: Observe the steady level of Mct-1 protein as cells are released from aphidicolin arrest. Middle: Cyclin E levels upon release demonstrate the expected decrease from peak levels at the G₁-S boundary. Bottom: Equal loading of the gels is confirmed by the B-actin blot. (b) Analysis of nuclear fraction for MCT-1 at different phases of the cell cycle. Jurkat cells were released from blockade and harvested at the indicated times. As shown, there is no detectable Mct-1 protein in the nuclear fraction. Controls included Cyclin B that translocates to the nucleus from the cytoplasm in a cell cycle related manner with peak levels in the nucleus at G2/M. Additional controls included PCNA, its nuclear expression reaching a maximum in S phase as indicated

Subcellular localization of MCT-1 remains constant throughout the cell cycle

The subcellular localization of some cell growth regulatory molecules have been shown to change depending on the phase of the cell cycle (Ye et al., 1998; Lam et al., 1997). After successful synchronization and release from G1/S block we analysed levels of MCT-1 protein in both nuclear and cytoplasmic fractions. As shown in Figure 2b, the results are similar to the earlier analysis in asynchronous cells with no detectable levels of MCT-1 in the nuclear fraction. Cyclin B was also examined since it has been previously shown to translocate to the nucleus from the cytoplasm in a cell cycle related manner with peak levels in the nucleus at G2/M (Figure 2b). Additional controls included PCNA, its nuclear expression reaching a maximum in S phase. Our results indicate that MCT-1 protein does not translocate to the nucleus in cycling cells.

MCT-1 displays a long half-life in human lymphoid cell lines

The protein synthesis inhibitor cyclohexamide was added to two T-cell lines, MT-2 and Jurkat at a concentration of 5 μg/ml and remained present until harvest time. As demonstrated in Figure 3, the half-life of MCT-1 in the MT-2 cell line is approximately 19 h. Similar results (not shown) were obtained in Jurkat cells. Controls included the p53 protein which is known to be stabilized in MT-2 cells and had a half-life of about 8 h as determined in our experiments consistent with published work (Reid et al., 1993; Gartenhaus and Wang, 1995).

Figure 3

Half-life determination of MCT-1. The protein synthesis inhibitor cyclohexamide was added to the MT-2 T-cell line at a concentration of 5 μg/ml and remained present until harvest time. Protein quantification was carried out using laser densitometry (Molecular Dynamics), and half-life determination was calculated using standard methodology. The half-life of MCT-1 in the MT-2 cell line is approximately 19 h. Controls included the p53 protein which is shown to be stabilized in MT-2 cells with a half-life of approximately 8 h. Virtually identical data were obtained in two independent experiments

MCT-1 expression is rapidly induced in X-irradiated human lymphoid cells but not to other stressful stimuli such as heat shock

In order to examine if MCT-1 protein levels were effected by exposure to DNA damaging agents, we exposed exponentially growing normal donor PBL or relevant T-cell lines to one of the following; ionizing radiation (IR) using a ¹³⁷Cs source, adriamycin or taxol. Whole cell lysates (30 μg total protein) were prepared at the indicated time points for Western blot analysis with the indicated antibodies. We observe the rapid increase of MCT-1 protein levels in normal donor lymphocytes as early as 2 h after exposure to these agents (Figure 4). This increase in MCT-1 protein appeared not to require induction of p53 as demonstrated in the taxol treatment arm (Figure 4). A moderate dose response effect to gamma irradiation was observed with increasing induction of MCT-1 protein levels as indicated in Figure 5a. Next, we examined two HTLV-1 transformed T-cell lines establishing that this DNA damage response is operative in both normal and transformed lymphocytes (Figure 5b). Finally, in order to address whether MCT-1 protein induction was a general response to stress stimulus we examined the cellular levels of MCT-1 after heat shock exposure. The Jurkat and EC155 T-cell lines were placed at 42°C for 45 min, protein lysates were prepared and examined by Western blot. As shown in Figure 6, there was induction of the control Hsp 27 in both cell lines but MCT-1 protein levels remained constant. Thus, it appears that general stress stimuli such as heat shock do not induce MCT-1.

Figure 4

MCT-1 expression is induced in response to DNA damaging agents and a microtubule-stabilizing agent. MCT-1 protein levels in normal donor lymphocytes after exposure to adriamycin (10 μM), taxol (1 μM) and gamma irradiation (10 Gy). Treated cells were harvested at the indicated time points after exposure. Observe the significant MCT-1 protein induction as early as 2 h post-treatment. Controls included the tumor suppressor p53 showing induction as expected after exposure to the DNA damaging agents but not after taxol, a microtubule-stabilizing agent. Experiments were independently performed at least four times with similar results

Figure 5

Normal human lymphocytes demonstrate a dose response induction of MCT-1 to gamma irradiation and this response is also seen in transformed T-cell lines. (a) PBL exposed to the indicated dose of IR (1–100 Gy). Cell lysates from treated cells and untreated controls were obtained 2 h post-treatment and examined for MCT-1, p53 and B-actin protein levels. Observe the increase in MCT-1 protein correlating with increasing IR exposure. (b) Western blot Analysis of EC155 and N1186 after treatment with 1000 rads (10 Gy). Cell lysates from treated cells and untreated controls were obtained 2 h post-treatment and examined for MCT-1, p53 and B-actin protein levels. Experiments were independently performed at least three times with similar results

Figure 6

MCT-1 expression is not induced by heat shock. In order to address whether MCT-1 protein induction was a general response to stress stimulus we examined the cellular levels of MCT-1 after heat shock exposure. The Jurkat and EC155 T-cell lines were placed at 42°C for 45 min, protein lysates were prepared and examined by Western blot. There was induction of the Hsp 27 in both cell lines though less so in the EC155 line. In both cell lines there was no induction of MCT-1 protein levels observed after heat shock treatment. Therefore, it appears that general stress stimuli such as heat shock do not induce MCT-1

The increase in MCT-1 protein expression in irradiated human lymphoid cells does not occur at the mRNA level, and does not require new protein synthesis

To investigate whether the induction of MCT-1 protein expression in response to gamma irradiation is due to altered transcript levels we pretreated control and gamma irradiated normal lymphocytes with actinomycin D, a transcriptional inhibitor at 10 μg/ml. After 1 h pretreatment, cells were exposed to 10 Gy and analysed for levels of MCT-1 protein. As shown in Figure 7, there was still significant induction of MCT-1 protein at 2 h post-treatment. The control p21/Waf protein which increases in response to gamma irradiation primarily through a p53 dependent transcriptional mechanism was completely uninduced confirming the successful block of transcription. We also measured MCT-1 mRNA levels in control and irradiated T-cells by Northern blot. There was no difference in mRNA levels post-irradiation (data not shown). These data exclude transcriptional/post-transcriptional regulation as a mechanism for the induction of MCT-1 in response to gamma irradiation. We next investigated whether new protein synthesis is required for the up-regulation of MCT-1 in response to gamma irradiation using the protein synthesis inhibitor, cyclohexamide. After pre-treatment with cyclohexamide (5 μg/ml) for 1 h, cells were irradiated and examined for induction of MCT-1. The data reveal an increase in the irradiated lymphocytes of MCT-1 but not of p21/Waf (Figure 7). The inhibition of induction of p21/Waf attests to the successful translational arrest due to cyclohexamide and that increased expression of MCT-1 protein must involve other mechanisms.

Figure 7

Altered MCT-1 protein expression in irradiated cells does not occur at the mRNA level and does not require new protein synthesis. We pretreated control and gamma irradiated normal lymphocytes with actinomycin D, a transcriptional inhibitor at 10 μg/ml. After 1 h pretreatment, cells were exposed to 10 Gy and analysed for levels of MCT-1 protein. At 2 h post-treatment there was significant induction of MCT-1 protein. The p21/Waf protein was completely uninduced confirming the successful block of transcription. We next examined if the protein synthesis inhibitor, cyclohexamide could block up-regulation of MCT-1 in response to gamma irradiation. After pre-treatment with cyclohexamide (5 μg/ml) for 1 h, cells were irradiated. The data reveal an increase in the irradiated lymphocytes of MCT-1 but not of p21/Waf. There was a modest increase after cyclohexamide alone in the level of MCT-1 protein but a significantly greater increase after gamma irradiation was observed. The inhibition of induction of p21/Waf confirms that cyclohexamide was biochemically active in the treated cells and that increased expression of MCT-1 protein must involve other mechanisms

Discussion

In previous work we screened a panel of T-cell leukemia/lymphoma lines using AP–PCR and identified an amplified DNA sequence in the Hut 78 lymphoid cell line (Prosniak et al., 1998). Expression analysis of a variety of normal human tissues revealed low level ubiquitous expression of MCT-1 (Prosniak et al., 1998). The MCT-1 cDNA sequence exhibited a high degree of evolutionary conservation across species (unpublished data). MCT-1 is a novel gene which when overexpressed in cells shortens the time to progress through G₁/S phase, decreases cell doubling time and confers a transformed phenotype (Prosniak et al., 1998). Recent work has established increased levels of MCT-1 protein in IL-2-independent T-cell leukemia/lymphoma cell lines relative to growth factor dependent T-cell lines (Dierov et al., 1999). In primary quiescent Chronic Lymphocytic Leukemia cells we found low to undetectable levels of MCT-1 protein consistent with their arrest in G₀/G₁ (manuscript in preparation). The present study has examined the expression level of MCT-1 throughout the cell cycle.

The expression of many cell cycle regulators including cyclins and cdk inhibitors vary according to the progression of the cell cycle. We consequently examined the cell cycle regulation of MCT-1 protein levels at different phases of the cell cycle. As shown here, lymphoid cells arrested at G₁/S, then released and allowed to progress through the cell cycle display little variation of MCT-1 protein during the cell cycle. The levels of cyclin E validate the successful synchronization as its levels decrease as expected upon release from G₁/S phase boundary blockade.

We examined the subcellular localization of MCT-1 protein in two exponentially growing lymphoid cell lines using differential centrifugation to separate the various cellular fractions. We demonstrated that the major subcellular localization of MCT-1 in asynchronous cell populations appears to reside in the cytosolic and light membrane fractions. It is highly reproducible in two distinct T-cell lines, HUT 78 and the HTLV-1 transformed MT-2.

To gain a better understanding of the possible mechanisms involved in MCT-1 function, we also studied its intracellular localization as a function of the cell cycle. The subcellular localization of some cell cycle regulatory molecules has been shown to change depending upon the cell cycle stage (Petersen et al., 1999). We therefore examined the subcellular localization of MCT-1 in T-cell lines that were synchronized at G₁/S phase boundary and released back into the cell cycle and analysed at various time points. Nuclear and cytoplasmic fractions were obtained (Poommipanit et al., 1999) for protein analysis. The cell cycle profile and MCT-1 localization were monitored by flow cytometry of propidium iodide-stained cells and Western blotting. In two independent T-cell lines we observed no translocation of MCT-1 to the nucleus throughout the cell cycle. These results are consistent with the lack of a nuclear localization signal in MCT-1. To verify the successful isolation of nuclei and cell cycle synchronization, we examined cyclin B since it has been previously shown to translocate to the nucleus from the cytoplasm in a cell cycle related manner with peak levels in the nucleus at G2/M. Analysis of PCNA further corroborated our findings with its nuclear expression peaking in S phase as previously reported.

In contrast to many cell cycle regulators, MCT-1 levels remain constant during the cell cycle in proliferating cells, suggesting that the activity of the protein is restricted by another mechanism(s). It may be that there are post-translational modifications of MCT-1 relative to the phase of the cell cycle. The MCT-1 protein is predicted to have numerous putative phosphorylation sites including one tyrosine site, two PKC sites and a consensus cyclinB/cdc2 kinase substrate site (Prosniak et al., 1998; unpublished data). Phosphorylation has been shown to be involved in the regulation of many cellular processes, including protein-protein interactions, protein stability, kinase activity and protein localization. Additional studies will be required to determine the factors regulating MCT-1 function during the cell cycle and to clarify its role in cell cycle progression.

Deregulation of the G₁-S checkpoint is associated with cells progressing through the cell cycle despite sustaining DNA damage. Both cyclin D1 and MCT-1 are involved in regulating passage of cells through the G1 phase of the cell cycle. A previous report has shown that gamma irradiation induces cyclin D1 expression (Epperly et al., 1995) In order to examine if MCT-1 protein levels were also modulated by exposure to gamma irradiation, we exposed exponentially growing normal donor PBL to ionizing radiation (IR) using a ¹³⁷Cs source and consistantly observed a 3–5-fold increase in MCT-1 protein. The dose of 10 Gy was chosen as this has reproducibly demonstrated induction of p53 in our T-cell lines as well as in normal PBL. It should be noted that the induction of MCT-1 was not as dramatic as induction of p53 or its downstream target p21. We extended these studies to include another DNA damaging agent, adriamycin. Similar results were obtained with induction of MCT-1 protein as early as 2 h after exposure. In order to determine whether this response was restricted to DNA damaging agents, we employed Taxol, a microtubule-stabilizing agent. Interestingly, there was a comparable amount of MCT-1 induction. The p53 tumor suppressor gene does not appear to be required for MCT-1 induction as illustrated in the taxol treatment arm where we observed no induction of p53. Furthermore, we demonstrated that MCT-1 induction was not simply a universal sensor of cellular stress as evidenced by our heat shock experiments. Since taxol works at clinically relevant doses primarily through its tubulin-binding properties (Chaung et al., 1994) and downstream serine phosphorylation of proteins including Bcl-2 (Blagosklonny and Fojo, 1999), our findings suggest potential avenues of research. For example, the MCT-1 protein has at least one potential serine phosphorylation site and will be examined for phosphorylation modifications both before and after treatment. Our experiments performed with actinomycin and cyclohexamide indicate that increases in MCT-1 protein in irradiated human lymphoid cells does not occur at the mRNA level and does not require new protein synthesis.

The cellular response to genotoxic stress is complex and it is unclear at present the role that MCT-1 plays in this response. A recently described RNA-binding domain, designated PUA domain (Aravind and Koonin, 1999) has been identified in the MCT-1 protein (Gastenhaus and Shi, manuscript in preparation). This novel domain has been associated with the translation machinery and has also been detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1 (Sheikh et al., 1999). It has been suggested that modulation of translation initiation may be an important adaptive response to genotoxic and other cellular stress (Sheikh et al., 1999). Further work is required in order to examine the role of MCT-1 in the cellular response to genotoxic stress and the specific function of the PUA domain.

The similar pattern of MCT-1 protein induction in response to two distinct stimuli i.e., ionizing radiation and a microtubule-stabilizing agent suggests alternative signaling pathways converging downstream. Our aggregate data support the hypothesis that induction of MCT-1 at least in response to irradiation is through increased stabilization by post-translational mechanism(s) unknown at present.

Materials and methods

T-cell lymphoid cell lines and peripheral blood lymphocytes

T-cell lymphoid tumors included both IL-2 dependent; EC155 (Advanced Biotechnologies, Columbia, MD, USA) NII85 and N1186 (Berneman et al., 1992) and IL-2 independent; Hut 78, MT-2 and Jurkat (Advanced Biotechnologies, Columbia, MD, USA). All cell lines were grown in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (Life Technologies, Inc) and IL-2 dependent T-cell lines were also supplemented with 40 μ/ml of recombinant IL-2 (Life Technologies, Inc). Normal donor peripheral blood lymphocytes (PBL) were centrifuged over Ficoll-Hypaque (Pharmacia, Pleasant Hill, CA, USA) isolating mononuclear cells. After PHA stimulation the lymphocytes were cultured in a similar manner as the IL-2 dependent T-cell lines.

Cell cycle synchronization

Fully confluent Jurkat cells were blocked at the G1/S interphase using 5 μg/ml aphidicolin for 18 h. Following a thorough washing of the cells they were subsequently resuspended in growth medium and harvested at the indicated time points for analysis of MCT-1 protein levels as a function of the cell cycle. In some instances we employed a double aphidicolin block. Briefly, logarithmically growing cells were blocked in S phase overnight with 1 μg/ml aphidicolin (Sigma), washed twice in complete RPMI 1640 and then released into fresh, prewarmed complete RPMI 1640. Following the completion of S phase, cells were again blocked overnight at the G₁/S border with 5 μg/ml of aphidicolin. Cells were washed three times and released into fresh, prewarmed complete media for time-course analysis. Cells from each time point were washed twice with ice cold PBS. An aliquot of these cells was fixed and stored in 70% ethanol at −20°C for cell cycle analysis, a second aliquot was used to make whole cell lysates, while the remaining cells were used to isolate nuclei. The viability of cells at the end of the time course was ⩾98% as determined by trypan blue exclusion. Fluorescence data were collected with the Coulter Epics XL-MCL flow cytometer and the percentage of cells within the G₁, S, and G₂-M phases of the cell cycle was determined by analysis with the software program MultiCycle (Phoenix).

Preparation of cellular fractions

Cells were lysed in hypotonic buffer (42.5 mM KCl, 5 mM MgCl₂, and 10 mM HEPES, pH 7.4) by passaging them four times through a 30-gauge needle. Isotonicity was reestablished by adding an equal volume of hypertonic buffer (242.5 mM KCl, 5 mM MgCl₂, and 10 mM HEPES, pH 7.4). Nuclei and unlysed cells were pelleted twice at 200 g for 10 min. The supernatant was centrifuged at 10 000 g for 10 min to collect the heavy membrane pellet. That supernatant was centrifuged at 100 000 g for 60 min, and the final supernatant was collected as the soluble fraction, and the pellet was collected as the light membrane fraction. The heavy membrane pellet was washed twice in H medium (0.25 mM mannitol, 0.075 M sucrose, 1 mM EGTA, 5 mM HEPES, pH 7.4, and 0.1% fatty acid-free bovine serum albumin). Both heavy and light membrane pellets were lysed in RIPA buffer and, together with the soluble fraction, were stored at −70° for immunoblotting.

Nuclear isolation

Cells for nuclear isolation were washed twice in hypotonic buffer (10 mM HEPES, pH 7.5, 5 mM KCl, 2 mM MgCl₂), resuspended in hypotonic buffer plus 1 mM PMSF, 0.5 mM DTT, and protease inhibitor cocktail (1 : 20, Sigma) and swelled on ice for 15 min. Nuclei were released by subjecting cells to 5–15 strokes with a Dounce homogenizer (pestle B), washed twice with hypotonic buffer, lysed in sample buffer, boiled for 5 min and clarified by centrifugation at 16 000 g for 15 min. The integrity and purity of nuclei for each preparation was verified by trypan blue and was ⩾99%.

Immunoblotting

Cells lysates were prepared for immunoblotting as follows: cells were washed twice in PBS, lysed in sample buffer (62.5 mM Tris, pH 6.8, 2% SDS) and boiled for 5 min. Sample viscosity was reduced by passage through a 20 ga needle and lysates were clarified by centrifugation at 16 000 g for 15 min. Protein concentrations were determined using the Bradford assay (BioRad, Hercules, CA, USA) and 20 μg of protein were separated on 12.5% gels by SDS–PAGE and transferred to Hybond ECL nitrocellulose (Amersham Pharmacia Biotech, Buckinghamshire, UK). Membranes were blocked in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20)+5% nonfat dried milk for 1 h at room temperature and then incubated with primary antibody diluted in block buffer for 1 h at room temperature. Blots were then washed three times (10 min each) with TBST and subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (sheep anti-mouse IgG or donkey anti-rabbit IgG, diluted 1 : 5000 in TBST (Amersham Pharmacia Biotech) or an anti-goat IgG diluted 1 : 7000 in block buffer (Santa Cruz, Santa Cruz, CA, USA)) for 1 h. Blots were again washed three times in TBST and specific proteins were detected by ECL (Amersham Pharmacia Biotech). Primary antibodies included a rabbit polyclonal anti-Mct-1 (Research Genetics), a mouse monoclonal anti-Bcl-2 (Santa Cruz), a rabbit polyclonal anti-Fas (Santa Cruz), a mouse monoclonal anti-β-actin (Sigma, St. Louis, MO, USA), a rabbit polyclonal anti-c-Myc (Santa Cruz), a goat polyclonal anti-NF-κB (Santa Cruz), a rabbit polyclonal anti-cyclin E (Santa Cruz), a mouse monoclonal anti- PCNA (Santa Cruz), a mouse monoclonal anti-p53 (Santa Cruz), a mouse monoclonal anti-Hsp27 (Santa Cruz) and a mouse monoclonal anti-cyclin B1 (Santa Cruz). Equal concentrations of protein from the various subcellular fractions were loaded unto gels with quantitation determined by using laser densitometry.

DNA damage and heat shock stress response

In order to examine if MCT-1 protein levels were effected by exposure to DNA damaging agents, we exposed exponentially growing normal donor PBL or relevant T-cell lines to ionizing radiation (IR) using a ¹³⁷Cs source (1–100 Gy) and harvested cells at the indicated time points for Western blot analysis, protein levels were quantitated by laser densitometry (Molecular Dynamics) beginning at 2 h after IR exposure. We also examined the effect of Adriamycin (10 μM) and taxol (1 μM) on cellular levels of MCT-1. The p53 protein was analysed to confirm adequate delivery of a DNA damage dose. In order to determine if heat shock exposure would induce MCT-1, the Jurkat and EC155 T-cell lines were placed at 42°C for 45 min and cell lysates were examined by Western blot.