Unscheduled cyclin B expression and p34 cdc2 activation in T lymphocytes from HIV-infected patients

Objective: To study the role of cell cycle regulation during HIV infection by investigating in vivo and in vitro cyclin B and p34 cdc kinase expression.

Methods: Cyclin B expression was analysed by Western blot in CD4 and CD8 cells from 25 HIV-infected patients and 24 uninfected individuals. In eight patients, a sequential analysis was performed after initiation of antiretroviral therapy (ART), and correlations with CD4 cell count and HIV viremia were studied. Sequential changes in cyclin B expression and p34 cdc kinase expression and activity were also studied in lymphocytes activated in vitro with phytohaemagglutinin (PHA).

Results: Lymphocytes from untreated HIV-infected patients demonstrate persistent in vivo overexpression of cyclin B in both CD4 and CD8 cell subpopulations. When cells are stimulated to proliferate in vitro, biochemical events that characterize the entrance into the cell cycle [ornithine decarboxylase (ODC) activity, interleukin 2 production, interleukin 2 a-chain receptor (IL-2R, CD25) expression, total protein synthesis, total DNA synthesis] show similar timing and sequence in lymphocytes from HIV-infected and uninfected individuals. However, in peripheral blood lymphocytes (PBL) from HIV-infected patients, cyclin B and p34 cdc kinase show premature expression during the cell cycle. Both in vivo cyclin B overexpression and in vitro unscheduled cyclin B expression were almost completely reversed 2-4 weeks after initiation of effective ART.

Conclusion: Increased and unscheduled expression of cyclin B and p34 cdc kinase is consistently observed in CD4 and CD8 cells from HIV-infected patients, both in vivo and after in vitro mitogenic stimulation. These alterations correlate with the level of viremia and may provide a link between the perturbation of lymphocyte proliferative homeostasis and the exaggerated propensity towards apoptosis.

Introduction

The hallmark of HIV-induced immunodeficiency is the progressive depletion of CD4 T lymphocytes, which indirectly causes the high rate of cell turnover aimed at replacing CD4 cells lost to viral killing [^1-3]. Moreover, HIV infection also induces a state of general immune activation in both blood and lymphoid organs, thus increasing the susceptibility of lymphocytes to programmed cell death (apoptosis). The origin of these alterations is complex and probably involves the loss of proper control of cell cycling and proliferation. More recently, the availability of highly active antiretroviral therapy (HAART) has provided key insights into the immunopathogenesis of HIV disease by allowing analysis of the reversion of some of the immunological changes related to the infection [^4-6].

The presence of cyclin B indicates whether a cell is cycling or quiescent. Programmed degradation of cyclins and consequent modulation of cyclin-dependent kinase (cdk) activity determine progression through the cell cycle, arrest in a particular phase of the cell cycle or death following receipt of a mitogenic signal [^7,8]. Cyclin B, the regulatory subunit of p34 cdc2 kinase, is synthesized and accumulates in complexes with the kinase during late G₁ and S phases [^7,9]. When p34 cdc2 kinase is activated, it catalyzes phosphorylation of different protein substrates such as nuclear lamins, vimentin and caldesmon [¹⁰]. These are necessary for disassembly and reorganization of the cytoskeleton during mitosis. To allow mitosis to proceed, the kinase must be rapidly inactivated and cyclin must be degraded by the proteasome [^8,11,12]. Persistent or untimely cdk activation is lethal to the cell because it is perceived as an apoptotic signal [^9,13,14]. Indeed, the tendency for peripheral blood lymphocytes (PBL) to undergo apoptosis in vitro is directly related to their position in the cell cycle in vivo. If cells are committed to cycle and then abruptly deprived of growth factors, they die [¹⁵].

When peripheral T lymphocytes express activation markers they are usually in proliferative phases of the cell cycle [¹⁶]. This state of immune/proliferative activation makes cells more susceptible to mitogen-induced apoptosis in culture, as was observed in both natural [¹⁷] and experimental [¹⁸] HIV infection. A correlation between immune activation and apoptotic intensity has also been reported in lymph nodes from HIV-positive patients [¹⁹].

We report here that perturbations of the lymphocyte proliferative homeostasis during chronic HIV infection are related to alterations of normal cell cycle control, and that an effective antiretroviral therapy (ART) is able to re-establish the proper control of cell cycle phases.

Materials and methods

Patient population

Twenty-five HIV-infected patients attending the service of Clinical Immunology at the University of Ancona were selected based on CD4 cell counts of >100¥10⁶ cells/l and lack of previous ART. Eight of these underwent a therapeutic regimen comprising zidovudine (Retrovir, Glaxo-Wellcome, Research Triangle Park, NC, USA; 200mg three times daily) plus didanosine (Videx, Bristol Myers Squibb, Princeton, NJ, USA; 200mg twice daily). All HIV-infected patients belonging to this study remained asymptomatic throughout the duration of the study. After obtaining informed consent, blood samples were collected, PBL isolated and intracellular concentrations of cyclin B and p34 cdc2 kinase activity measured at baseline and at least eight times during the first 6 weeks after the initiation of therapy; at these times, all patients treated by ART also underwent laboratory testing and clinical examination, as described previously [²⁰]. In order to assess spontaneous fluctuation of cyclin B and p34 cdc intracellular concentrations in PBL, the same sequential analysis was performed in four HIV-infected patients undergoing chronic ART and in two uninfected controls.

Immunological phenotyping was performed on FACScan (Becton Dickinson, San Jose, CA, USA) by staining cells with the following antihuman monoclonal antibodies: CD4, CD8, HLA-DR, CD95, CD38 and CD28 [²⁰]. Twenty-four uninfected individuals (including five drug abusers) were enrolled to the study as controls.

Cells

CD4 and CD8 T cells were purified and cultured from PBL [^17,18,21]. Cell cycle timing (G₁ and S phase) was established by measuring the following cell metabolic parameters: ornithine decarboxylase (ODC) activity and membrane transport of small metabolites, including glucose and amino acids (proline) (transition from G₀ to G₁ phase); expression of interleukin 2 (IL-2) a-chain receptor (IL-2R, CD25) and synthesis of IL-2 (early G₁); and activity of cell machinery for protein (late G₁) and DNA synthesis (S phase) [^22,23].

Cyclin B and p34 cdc2 intracellular concentrations were measured by Western blot (monoclonal antibodies from Santa Cruz Biotechnology Inc., Santa Cruz, CA); the bands in the gel image were analysed using SigmaGel (Jandel Scientific Software Co., San Rafael, CA, USA). The numerical values, on a scale from 0 to 250, indicate the absolute area of the band, i.e., the total calibrated pixel intensity values of each band. Between two and five replicates were performed for each patient and control.

In all measurements of cyclin B and p34 cdc intracellular concentrations, several internal controls were always performed, including lysing equal cell numbers, determining the amount of loaded proteins by the Bradford method, loading into each lane equal volumes of equal protein concentrations (15mg/lane), and, after the electrophoresis, by performing Coomassie brilliant blue staining. If different protein concentrations were observed at this time, the whole procedure was repeated and the initial loading protein concentration adjusted on the basis of the actin band.

P34 cdc2 kinase activity has been measured as phosphorylation of H1 histone by p34 cdc2 SPA kinase enzyme assay kit from Amersham International plc (Little Chalfont, UK). Viral load has been measured as detailed in [²⁰].

Results

To study potential alterations of cell cycle control induced by HIV infection, the expression of cyclin B in lymphocytes from HIV-infected individuals was investigated by Western blot analysis. Freshly isolated lymphocytes from HIV-infected patients showed intracellular cyclin levels that are four- to ninefold higher than those in lymphocytes from healthy individuals. This increase was observed in sorted subpopulations of both CD4 and CD8 T lymphocytes (Fig. 1). Interestingly, between 2 and 4 weeks after the initiation of ART with zidovudine and didanosine, cyclin B expression decreased dramatically to levels comparable with those observed in lymphocytes of uninfected patients (Fig. 2). This decrease was preceded by a fall in viremia expressed as HIV RNA copies and followed by (i) an increase in the number of peripheral blood CD4 cells [²⁰]; (ii) a decrease in CD8+CD38+, CD8+CD95+ and CD8+HLA-DR+ cells; and (iii) an increase in the number of CD8+CD28+ cells [²⁴].

Fig. 1

Fig. 2

To examine the precise functional meaning of the constitutive cyclin B overexpression during HIV infection, the relationship between cyclin B expression, p34 cdc activity and the different phases of the cell cycle that follow in vitro activation of PBL was investigated.

Many biochemical events that characterize the entrance of resting (G₀) lymphocytes into the cell cycle following mitogenic stimulation, such as ODC activity, IL-2 production, IL-2R expression (early G₁ phase), proline uptake, total protein synthesis (late G₁ phase) and total DNA synthesis (S phase), showed similar timing and sequence in lymphocytes from both HIV-infected and uninfected individuals (Fig. 3). Although the absolute values of protein and DNA synthesis differed in healthy and infected individuals, both processes initiated and reached their maximum value at the same time following mitogenic stimulation of lymphocytes. These experiments clearly showed that PBL from HIV-infected patients retain the ability to enter the cell cycle effectively and to engage in all the biochemical events characteristic of the passage into phases G₁ and S.

Fig. 3

However, when the expression of cyclin B was studied after in vitro activation of lymphocytes from HIV-infected individuals, an obvious unscheduled overexpression of cyclin B was observed throughout the cell cycle. Furthermore, under the same conditions p34 cdc2 kinase activity was also constantly increased before, during and after S phase (Fig. 4). Conversely, in control cells and in cells from patients treated with ART, both cyclin B and p34 cdc kinase levels behaved as expected, abruptly increasing immediately before the cell enters the S phase and then declining at the end of the S phase (Fig. 4).

Fig. 4

Discussion

Overall, two major changes were observed in cyclin B expression in T lymphocytes from HIV-infected patients. First, cyclin B was overexpressed in resting lymphocytes (Figs 1 and 2) and, second, cyclin B expression was unscheduled during the cell cycle (Fig. 4). Cyclin B levels and activation-induced expression returned to normal following ART and the marked decrease in viral replication (Figs 2 and 4).

Cyclin B overexpression during HIV infection may result from increased synthesis or from reduced protein degradation, indicating, respectively, an increased number of cycling cells or a dysregulation of cell cyle control in presently resting cells that have already gone through multiple replicative cycles. In our experimental conditions, cyclin B accumulated in lymphocytes before the cell cycle-dependent machinery for protein synthesis was activated. When protein synthesis resumed following mitogenic stimulation of cultured cells, no relevant differences were observed in total cellular protein synthesis in controls and HIV-infected patients (from 0 to 1.9nmol in controls and from 0 to 1.7nmol in patients, expressed as leucine incorporated in 30 minutes by 10⁶ cells), although cyclin B remained constantly overexpressed in lymphocytes from HIV-infected patients. Overall, these findings suggest that the increased level of cyclin B in lymphocytes from HIV-infected patients may be a consequence of reduced protein degradation rather than increased synthesis. This, in turn, indicates that a more complex dysregulation of cell cycle control, rather than a pure increase in the rate of cycling cells in peripheral blood, is likely to be responsible for the increased intracellular concentration of cyclin B.

This hypothesis is confirmed by the finding that absolute values of p34 cdc2 activity are not much higher in T lymphocytes from HIV-infected patients than in controls (Fig. 4), yet the kinase is activated largely before S phase. The lack of cyclin B degradation seems to be directly responsible for this untimely activation of p34 cdc2 kinase, while excessive synthesis of cyclin B should be excluded as a cause of its overexpression. In fact, the intracellular concentration of p34 cdc2 kinase is comparable in HIV-infected patients and controls in all phases of the cell cycle (data not shown). These observations are in agreement with the report that high cyclin B intracellular levels are associated with premature activation of p34 cdc2 kinase and apoptosis of T cells [^25,26] and with the report that cyclin overexpression or unscheduled expression in neoplasms results from alterations in ubiquitin- mediated cyclin proteolysis [²⁷]. High intracellular levels of cyclin B also correlate with cell death during acute in vitro infection of CD4 cells, thus showing that HIV-induced single cell killing involves a block of cells in the G₂ phase of the cycle [²⁸].

In PBL from HIV-infected patients, cyclin B levels correlate with viremia, as shown by the effect of ART (Fig. 2). This observation suggests that HIV replication is indirectly responsible for an increase in peripheral blood in the number of cells that either are cycling or, more likely, have recently gone through multiple rounds of cell replication without an effective degradation of the previously synthesized cyclin B. Both of these events may be correlated with accelerated turnover or with a selective release of such cells from lymphoid tissue. The decrease in viremia resulting from anti-HIV therapy either induces a parallel decrease in the rate of cell turnover (and, therefore, decreases the number of cycling cells) or somehow corrects the dysregulation of cell cycle control that is likely to play a relevant role in the accumulation of cyclin B.

Our observation of cyclin B overexpression in vivo and accumulation of cyclin B with untimely p34 cdc2 activation in vitro may also provide an explanation for how the loss of cell cycle control and consequent increase in apoptosis are related in HIV disease. Several different apoptotic pathways have been described, and different cdk act as effectors in some of these [^25,26,29,30]. Apoptosis caused by premature p34 cdc2 activation, defined as mitotic catastrophe, is caused by a lack of coordination between phosphorylaton and degradation of specific protein substrates at critical points during mitosis [^{10,13,14,25,26}].

In conclusion, it is likely that the massive cellular activation/replication that takes place in the T cell compartment during chronic HIV infection with high levels of viremia leads to the continuous production of lymphocytes that have lost cell cycle control and are directed towards apoptosis mediated by cyclin B overexpression and premature activation of p34 cdc2 kinase. Furthermore, an effective anti-HIV therapy, which is known to reduce the activation state of PBL, also causes the cyclin B expression to return to normal levels in both freshly isolated and in vitro activated lymphocytes from HIV-infected patients.

Acknowledgments

The authors wish to thank Drs Mark B. Feinberg and Sara Perlaky-FitzGerald for helpful discussions.