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Keywords:

  • pituitary tumour transforming gene;
  • expression;
  • regulation;
  • human T cells;
  • cell cycle

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression of PTTG mRNA in T-lymphocyte activation
  6. Effect of immunosuppressants on PTTG expression
  7. PTTG expression in Jurkat T-cell line
  8. Discussion
  9. Acknowledgments
  10. References

Summary. Pituitary tumour transforming gene (PTTG) isolated from pituitary tumour cells transforms cells in vitro and causes in vivo tumour formation. PTTG is expressed in several human tumours and cell lines. In normal adult tissues, the testis expresses abundant levels of PTTG mRNA comparable to that found in tumour cells. Although PTTG is not expressed in resting T cells, we showed here that activation of normal adult human T cells using either immobilized anti-CD3 antibodies or phytohaemagglutinin was accompanied by marked PTTG induction, reaching levels observed in human tumour cells. Inhibitors of T-cell functions, such as cyclosporin A and hydrocortisone, decreased induction of PTTG mRNA expression. During T-cell activation, PTTG mRNA abundance corresponded with the increase in S-phase cells, suggesting PTTG involvement in cell cycle-dependent processes. These results showed that PTTG-1 expression follows cell cycling patterns in T lymphocytes, providing a convenient model for studying PTTG functions in normal cells.

Pituitary tumour transforming gene (PTTG) is highly expressed in pituitary tumours and other neoplasms, including the colon (Dominguez et al, 1998; Saez et al, 1999; Zhang et al, 1999a,b; Heaney et al, 2000). Levels of PTTG expression positively correlate with pituitary tumour invasiveness (Zhang et al, 1999b) and are induced by oestrogen (Heaney et al, 1999). In tumour cells, PTTG mRNA and protein expression are cell cycle dependent and peak at G2/M phase (Ramos-Morales et al, 2000; Yu et al, 2000a). Mechanisms for PTTG action have not yet been determined. PTTG upregulates basic fibroblast growth factor secretion (Heaney et al, 1999), trans-activates DNA transcription (Dominguez et al, 1998; Wang & Melmed 2000a) and stimulates oncogene c-myc expression (Pei, 2001). PTTG overexpression is associated with aneuploidy (Yu et al, 2000b).

PTTG is homologous to securin, which maintains the binding of sister chromatids during mitosis (Zou et al, 1999). At the end of metaphase, securin is degraded by an anaphase-promoting complex, releasing tonic inhibition of separin, which in turn mediates degradation of cohesins, the proteins that hold sister chromatids together. Overexpression of a non-degradable PTTG disrupts sister chromatid separation (Zou et al, 1999), and overexpression of PTTG causes apoptosis and inhibits mitoses (Yu et al, 2000b). The securin function of PTTG suggests that PTTG may also be expressed in normal proliferating cells. In adult animals and humans, PTTG mRNA is most abundant in the testis (Pei & Melmed, 1997; Zhang et al, 1999a).

Upon confronting an antigen, T cells rapidly proliferate from a quiescent state, thus providing a useful model to test PTTG function in normal cells. We studied the role of PTTG in T-cell proliferation and found that PTTG is strongly upregulated upon T-cell activation. Thus, normal adult human T cells could be a convenient model for the study of PTTG physiological functions and regulation in a non-transformed cell type.

Cell culture.  T lymphocytes were prepared by positive selection of mononuclear cells from fresh peripheral venous blood obtained from healthy human adult subjects. For some experiments, leucopack preparations of American Red Cross anonymous donors were used. Mononuclear blood cells were isolated by gradient centrifugation, using LymphoprepTM kit (Nycomed Pharma AS, Oslo, Norway). When leucopack preparations were used, isolated mononuclear cells were first frozen in 90% fetal bovine serum (FBS) supplemented with 10% dimethyl sulphoxide (DMSO). These cells were thawed and washed, and grown in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% FBS. Human Jurkat leukaemia T cells were grown in the same medium. To achieve Jurkat T-cell synchronization at the G1/S border, double thymidine block was performed (Yu et al, 2000a).

T-cell isolation and treatment. Double T-cell selection was performed using immunomagnetic beads (Dynal CD2 TM CELLectionTM kit; Dynal AS, Oslo, Norway) and cells were further activated by culturing in Petri dishes containing immobilized anti-human monoclonal CD3 antibody (Becton-Dickinson, Franklin Lakes, NJ, USA). Antibody immobilization was achieved by 90 min incubation (37°C) of fresh 60 mm plastic Petri dishes filled with 1 ml of anti-CD3 solution (10 µg/ml) in phosphate-buffered saline (PBS). Kit protocols provided by the manufacturers were used during T-cell isolation and activation. In some experiments, resting T cells were activated using phytohaemagglutinin (PHA, 5 µg/ml; Gibco-BRL, Grand Island, NY, USA). Relative amounts of anti-CD3-labelled T lymphocytes were checked after cell labelling with triple fluorochrome-labelled anti-CD3/CD19/CD45 antibodies (Caltag Laboratories, Burlingame, CA, USA) and flow cytometry (FACScan; Becton-Dickinson). Samples containing 95–97% of T cells and 0–0·05% of B cells were used in the experiments. Cells were also treated with the hydrocortisone 21-hemisuccinate (20–2000 nmol/l; Sigma, St Louis, MO, USA), cyclosporin A (0·1–2·0 µg/ml; Calbiochem, San Diego, CA, USA), transforming growth factor β1 (TGF β1, 10 ng/ml; R & D Systems, Minneapolis, MN, USA), aphidicolin (1 µg/ml; Calbiochem, San Diego, CA, USA) and nocodazole (500 ng/ml; Calbiochem). Control experiments for vehicles [0·1% C2H5OH (aphidicolin) and 0·2% DMSO (nocodazole)] did not alter PTTG mRNA expression.

Northern blot analysis. Total RNA was isolated using TRIzol reagent (Gibco-BRL). RNA (5 µg) was subjected to electrophoresis in 1% agarose–formaldehyde gel and blotted onto Hybond-N membranes (Amersham International, Little Chalfont, UK). Membranes were ultra-violet (UV) cross-linked, prehybridized for 1 h at 68°C in QuickHyb solution (Stratagen, La Jolla, CA, USA), hybridized for 3 h at 68°C in the same solution supplemented with random-primed 32P-labelled human PTTG or c-myc cDNA (2 × 106 cpm/ml) and sonicated with denaturated salmon sperm DNA (Stratagen). When interleukin 2 (IL-2) mRNA expression was studied, sequences of oligonucleotide sense and antisense primers for polymerase chain reaction (PCR) preparation of an IL-2 cDNA probe, sized 457 bp, were as previously reported (Butch et al, 1993). The cyclophilin cDNA probe was from Ambion (Austin, TX). Membranes were washed twice (20 min each time) in 1 × saline–sodium citrate (SSC) and 0·1% sodium dodecyl sulphate (SDS) at room temperature, followed by 30 min in 0·2 × SSC and 0·1% SDS at 60°C, and overnight exposure to radiographic film (Kodak, Rochester, NY, USA).

Fluorescence-activated cell sorting (FACS) analysis.  To study cell cycle patterns, cells were pelleted by centrifugation, washed in cold (4°C) PBS, re-suspended in 1 ml PBS and then 2 ml cold methanol slowly added for cell fixation. Propidium iodide (500 µl) (500 µg/ml PBS) together with 1 µl of ribonuclease A (10 units/µl) were added and cell suspension vortexed. After incubation for 1 h at 4°C, cell samples were analysed by flow cytometry. Analysis regions contained a minimum of 10 000 events. Cell cycle analysis was performed using modfit software, using a Macintosh computer.

Cell labelling with anti-CD3/CD19/CD45 antibodies.  Cell labelling with triple mouse anti-CD3/CD19/CD45 fluorochrome-labelled antibodies was performed using the manufacturer's protocol (Caltag Laboratories, Burlingame, CA, USA). Briefly, 15 µl Caltag triple antibody was added to 100 µl cell suspension, and 180 µl PBS added and gently mixed. Tubes were incubated at room temperature for 15 min in the dark, cells were fixed using Cal-Lyse lysing solution (Caltag Laboratories), washed with 3 ml de-ionized water, pelleted by centrifugation and re-suspended in 1 ml cold PBS. Flow cytometry was done using Becton-Dickinson FACScan.

Statistical analysis.  All experiments were repeated at least three times and the results of a typical experiment presented. Differences were assessed by Student's t-test with P < 0·05 considered significant. Non-linear regression analysis was performed for cyclosporin A and hydrocortisone effects on cell cycling. Non-linear regression analysis, based on χ2 criterion, was performed for cyclosporin A and hydrocortisone effects on PTTG mRNA expression and cell cycling in PHA-stimulated T lymphocytes. It revealed dose-dependent inhibition of studied indicators, which can be presented as a function:

  • image

c, cyclosporin A (or hydrocortisone) concentration; y0, A, t: different function coefficients.

Expression of PTTG mRNA in T-lymphocyte activation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression of PTTG mRNA in T-lymphocyte activation
  6. Effect of immunosuppressants on PTTG expression
  7. PTTG expression in Jurkat T-cell line
  8. Discussion
  9. Acknowledgments
  10. References

PTTG mRNA expression was studied in human adult T cells activated by anti-CD3 antibody and compared with those of c-myc and IL-2 (Fig 1), which are upregulated upon T-cell activation by mitogen anti-CD3. PTTG induction was observed 24 h after the start of mitogen action while c-myc induction was seen as soon as 2 h after T-cell activation and reached maximal levels by 24 h. IL-2 expression first increased 6 h after CD3 antibody application, while another, much more pronounced IL-2 expression occurred at 48 h (Fig 1A and B).

image

Figure 1. Time dependence of expression of mRNA for PTTG, c-myc and IL-2 in normal adult human T cells treated with mitogen CD3 antibody. Isolated T lymphocytes (5 × 106 cells suspended in 3 ml 10% FCS-supplemented RPMI-1640 culture medium in 35 mm dish) were stimulated with anti-CD3 antibody immobilized on the dish as described in Materials and methods. mRNA for PTTG, c-myc, IL-2 and 18S RNA were measured by Northern blotting of 5 µg total RNA per lane (A and B) and percentage of cells in S or G2/M phase determined by FACS (C). *P < 0·05; **P < 0·01; ***P < 0·001 (versus control: untreated cells).

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PTTG mRNA levels correlated with changes in number of cells in S phase and G2/M phase. PTTG mRNA expression was not readily detectable in resting T cells but dramatically increased as the percentage of cells in S and G2/M phase increased (48–72 h) (Fig 1C). No increase in the number of T cells in S or G2/M phase was observed 24 h after mitogen treatment. Thus, early induction of c-myc (2 h) and IL-2 (6 h) suggested that transcription of these genes was dependent on T-cell activation and not necessarily on proliferation.

After resting T cells were stimulated with another mitogen (PHA), a similar time course was seen for both T-cell proliferation and PTTG expression (Fig 2). T-cell proliferation and PTTG expression after 3 d of PHA stimulation were similarly dependent on PHA concentrations, up to 5 µg/ml (Fig 3). Higher concentrations of PHA further increased the number of cells in S phase, but decreased G2/M and had no further effect on PTTG expression.

image

Figure 2. Time dependence of PTTG mRNA expression in normal adult human T cells treated with phytohaemagglutinin. Isolated T cells (5 × 106 cells suspended in 3 ml 10% FCS-supplemented RPMI-1640 culture medium in 35 mm dish) were stimulated with PHA (5 µg/ml) for up to 72 h. PTTG mRNA and 18S RNA were measured by Northern blotting of 5 µg total RNA per lane (A) and percentage of cells in S or G2/M phase determined by FACS (B). *P < 0·05; **P < 0·01 (versus control: untreated cells).

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image

Figure 3. Dose dependence of PTTG mRNA expression in normal adult human T cells treated with phytohaemagglutinin. Isolated T cells (5 × 106 cells suspended in 3 ml 10% FCS-supplemented RPMI-1640 culture medium in 35 mm dish) were stimulated with PHA (up to 20·0 µg/ml) for 72 h. PTTG mRNA and 18S RNA were measured by Northern blotting of 5 µg total RNA per lane (A) and percentage of cells in S or G2/M phase determined by FACS (B). *P < 0·05; **P < 0·01 (versus control: untreated cells).

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Effect of immunosuppressants on PTTG expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression of PTTG mRNA in T-lymphocyte activation
  6. Effect of immunosuppressants on PTTG expression
  7. PTTG expression in Jurkat T-cell line
  8. Discussion
  9. Acknowledgments
  10. References

The immunosuppressants cyclosporin A (Fig 4A) and hydrocortisone (Fig 4B) inhibited PHA-stimulated T-cell proliferation and PTTG expression. Hydrocortisone action was slightly stimulatory at low doses and was inhibitory at concentrations higher than 100 nmol/l. Although cyclosporin A inhibited PTTG expression in human leukaemia Jurkat T cells, hydrocortisone did not alter PTTG expression in those cells (data not shown).

image

Figure 4. PTTG mRNA expression and the action of immunosuppressants: cyclosporin A (A) and hydrocortisone (B). Isolated normal adult human T cells (5 × 106 cells suspended in 3 ml 10% FCS-supplemented RPMI-1640 culture medium in 35 mm dish) were treated with PHA (5 µg/ml) and increasing doses of cyclosporin A (A) or hydrocortisone (B) for 72 h. PTTG mRNA and 18S RNA were measured by Northern blotting of 5 µg total RNA per lane (A) and percentage of cells in S or G2/M phase was determined by FACS (B). Non-linear regression analysis revealed a decrease of the studied indicators when treated with cyclosporin A and hydrocortisone.

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PTTG expression in Jurkat T-cell line

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression of PTTG mRNA in T-lymphocyte activation
  6. Effect of immunosuppressants on PTTG expression
  7. PTTG expression in Jurkat T-cell line
  8. Discussion
  9. Acknowledgments
  10. References

Jurkat T cells grew well in 10% FBS-supplemented RPMI-1640 medium without anti-CD3 or PHA stimulation. The abundance of PTTG mRNA in growing Jurkat cells was comparable to that in T cells activated by PHA or anti-CD3 antibody (Fig 5). PTTG expression decreased in Jurkat cells blocked at the G1/S border.

image

Figure 5. PTTG mRNA expression in normal adult human T cells and human leukaemia Jurkat T cells. Jurkat T cells (5 × 106 cells per 35 mm dish) were stopped at the G1/S border as described in Materials and methods, or grown in normal medium (cycling). Normal T cells (5 × 106 per 35 mm dish) were either kept quiescent, or treated with PHA (5 µg/ml) or CD3 antibody for 72 h. PTTG mRNA and 18S RNA were measured by Northern blotting of 5 µg total RNA per lane.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression of PTTG mRNA in T-lymphocyte activation
  6. Effect of immunosuppressants on PTTG expression
  7. PTTG expression in Jurkat T-cell line
  8. Discussion
  9. Acknowledgments
  10. References

In this study, we demonstrated that PTTG expression was enhanced in activated T lymphocytes. PTTG expression correlated with the percentage of cells in S and G2/M phase, consistent with previous findings in a tumour cell line that PTTG expression is cell cycle dependent and peaks at G2/M phase (Yu et al, 2000a). Cyclosporin A and hydrocortisone inhibited PTTG expression, further supporting that PTTG expression was upregulated in T-cell proliferation. Thus, regulation of PTTG expression in tumour cell lines appeared to be similar to normal proliferating T cells. Although increased PTTG mRNA could be attributed to increased transcription or decreased degradation, it is probably the former mechanism that acts in proliferating T cells. The human (Kakar, 1999) and mouse (Wang & Melmed, 2000b) PTTG promoters contain several highly homologous motifs, including the CCAAT, CDE (cell cycle-dependent element) and CHR (cell cycle homology region), which are present in other cell cycle-dependent genes.

IL-2 expression is an early activated gene specific for T cells (Kronke et al, 1984; Crabtree & Clipstone, 1994). Increased IL-2 mRNA expression in T cells occurred as early as 6 h after mitogen stimulation, and preceded PTTG mRNA expression and cell proliferation. Cyclosporin A exerted its inhibitory action on T lymphocytes via IL-2 gene suppression, leading to inhibition of T-cell DNA synthesis (Kronke et al, 1984; Crabtree & Clipstone, 1994). Thus, IL-2 may participate in the induction of PTTG expression.

c-myc is an immediate early gene, and PTTG binds the c-myc promoter and activates c-myc transcription (Pei, 2001). Here we have shown rapid (2 h) c-myc expression and much later (24 h) PTTG expression in normal human adult T cells subjected to mitogen CD3 antibody action.

In summary, we showed PTTG induction during T-cell proliferation. Thus, normal adult human T cells are a novel, convenient model for studying PTTG physiological functions in non-transformed cells.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression of PTTG mRNA in T-lymphocyte activation
  6. Effect of immunosuppressants on PTTG expression
  7. PTTG expression in Jurkat T-cell line
  8. Discussion
  9. Acknowledgments
  10. References

The authors are grateful to Dr Sandra McLachlan and Dr Basil Rapoport for helpful discussions and critical reading of the manuscript, Patricia Lin for FACS analysis, Gregory Horwitz for PTTG cDNA-containing plasmid, Loredana Farilla for IL-2 cDNA, Dr Pavel Pichugin for technical assistance, and Rostyslav Bilyi for help in figure preparation and statistical analysis. This work was supported by NIH grant CA 75979 and the Doris Factor Molecular Endocrinology Laboratory.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression of PTTG mRNA in T-lymphocyte activation
  6. Effect of immunosuppressants on PTTG expression
  7. PTTG expression in Jurkat T-cell line
  8. Discussion
  9. Acknowledgments
  10. References
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