Pre T-Cell Receptor Alpha (pTα) Expression Patterns and Functional Analysis in Human T-Cell Lymphoblastic Leukemia

Background: The pTα/preTCR regulates the β-selection, a crucial T-cell developmental checkpoint, providing a most potent survival advantage to thymocytes mediated by the src-kinase p56Lck. Methods: To define the relevance of pTα in human T-cell lymphoblastic leukemia (T-ALL), we analyzed in T-ALL cell lines (n=14) pTα and p56Lck mRNA and protein expression as also the tyrosine-phosphorylation. The p56Lck specific src-protein-tyrosine kinase inhibitor (PTK-I) PP1 was used in growth inhibition assays. IC50 value determination, cell cycle- and apoptosis analyses were performed in T-ALL-, non-T-ALL- and murine transgenic cell lines. Results: pTα expression patterns were markedly different in T-ALL cell lines as compared to those reported for normal lymphoid counterparts. PP1 induced in 6/11 T-ALL cell lines a survival disadvantage resulting from a cell cycle arrest in the G1/0 phase in thymic lymphoblastic cells and apoptosis induction in the immature cell line HSB-2, respectively. PP1 sensitive cell lines expressed the target protein p56Lck and showed a corresponding P-Tyr signal. Conclusions: Sensitivity of thymic T-ALLs to PP1 clearly underlines the impact of pTα mediated proliferation in this leukemic sub-type. In addition, p56Lck represents also independently of pTα a promising therapeutical target for the src-kinase inhibitors in neoplastic lymphoid diseases.


Background
Emergence of T-ALL is thought to result from specific interference of T-cell oncogenes with develop-mental networks regulating proliferation, survival and differentiation [10]. However, T-ALL clones retain characteristics of lymphoid precursors as illustrated by a shared expression pattern of surface molecules [4].
While the relevance of pTα/preTCR expression in leukemogenesis is currently a disputed topic, leukemogenesis in Notch3-ICN transgene mice showed a striking dependence on pTα expression [1,3,7,20,27]. Additionally, the interpretation of the functional relevance of pTα/preTCR in human T-ALL cells is controversial due to the lack of antibodies, low surface expression and novel insights that implicate pTα itself, independently of other components of the preTCR as the mediator of this early survival signal [26].
Here, we analyzed pTα gene-and protein-expression patterns and employed the src-PTK-I PP1 to study the biological relevance of the pTα/preTCR in human T-ALL cell lines.

Materials and methods
Cell lines (n = 18) were obtained from the DSMZ (Braunschweig, Germany, http://www.dsmz.de). T-ALL cell lines (n = 14) were classified according to the EGIL-criteria [4], while pre-and pro-T-ALL due to the low number of available cell lines were categorized as immature T-ALL. Murine SCB.29 and SCBγ/δ.28 were provided by H. von Boehmer (Dana-Faber Cancer Institute, Boston, MA, USA) [17]. pTα a (wild-type) expression was quantified by realtime RT-qPCR and normalized to β-actin. RT-PCR was performed for: pTα b (truncated isoform), β-actin and p56 Lck PCR conditions and primer sequences can be obtained from the corresponding author.
Western blot analysis of the pTα-chain (murine monoclonal antibody 2F5, Hybridoma J2.3, obtained by H. von Boehmer, Boston, MA, USA) and actin (murine monoclonal antibody, C2, Santa Cruz Biotechnology, CA, USA) was accomplished according to standard protocols, using equal protein lysate amounts (3 × 10 5 cells; n = 20; each sample was tested at least twice) [16]. Best signals were used for further normalization to the actin signal and semiquantification of pTα protein expression was performed using scion image beta (V. 4.02, USA). Prior to analysis of the cell lines, the specificity of the antibody 2F5 was validated in a competitive antigen assay using crystallized pTα antigen (kindly provided by M. Probst-Kepper, Helmholtz-Center for Infection Research, Braunschweig, Germany) and β 2 -Microglobulin (Santa Cruz Biotechnology, CA, USA). p56 Lck protein (rabbit monoclonal antibody, 73A5, Cell Signaling, Boston MA, USA) and total cellular tyrosine phosphorylation (murine monoclonal anti-body, P-Tyr-100, Cell Signaling, USA) were analyzed in equal cell lysate amounts (3 × 10 5 cells; testing the effect of PP1: 1.5 × 10 5 cells) by Western blotting according to standard protocols (n = 15; analyzed at least twice) [12,16]. β-actin was used as a loading control (monoclonal mouse antibody, AC-47, Sigma, Germany). The bound antibodies were detected by horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence (Santa Cruz Biotechnology, CA, USA and Cell Signaling, USA). Summarizing heat maps were compiled for quantitative pTα a -, semi-quantitative pTα band p56 Lck -mRNA and pTα protein expression with Genesis (V. 1.7.2, A. Sturn, Graz University of Technology, Austria) [19].

Results and conclusion
Analysis of peripheral blood mononuclear cells (PBMCs) from T-ALL patients at disease onset and in remission identified combined expression of pTα a , its isoform pTα b and Notch3 of particular impor-tance for sustaining T-cell leukemogenesis, potentially representing a pathognomonic molecular signature of human T-ALL [3]. In contrast to this finding but in agreement with Asnafi et al. [1], we detected mRNA expression of pTα isoforms in patients with T-ALL, B-ALL, AML and also in healthy controls (data not shown). Expression analysis of pTα in PBMCs from T-ALL patients might be misleading since pTα expression in these specimens is most likely not originating from the leukemic population, but rather from CD4 + CD3 − extrathymic T-cell progenitors [6], whose characteristics in T-ALL are unknown. To avoid this bias, our analyses were performed in homogenous T-ALL cell lines.
Among all analyzed T-ALL subtypes, immature T-ALLs showed the highest average pTα a mRNA expression, and pTα b mRNA was found in two out of three immature T-ALLs (Fig. 1A, B). pTα protein was detected in three out of three immature T-ALLs (Fig. 1D). Intermediate pTα a mRNA expression was found in thymic T-ALLs, and pTα b was detected in most of these cells (Fig. 1A, B). pTα protein was detected in six out of six thymic T-ALLs (Fig. 1D).
Mature T-ALLs expressed the lowest level of pTα a mRNA, and only two out of four mature T-ALLs expressed pTα b (Fig. 1A, B), whereas all mature T-ALLs expressed pTα protein (Fig. 1D).
Physiological pTα isoform expression is differentially regulated during T-cell maturation [2,15]. Nor-mally, pTα a expression increases until β-selection onset, while the transcriptional onset of pTα b is delayed, peaking after the β-selection and potentially regulating a rapid loss of pTα a after β-selection, thus terminating the cellular and molecular effects of pTα [2,15]. During the thymic window, where the β-selection is supposed to occur, pTα b is believed to strengthen the pTα a signal [2,15]. Quantitative and qualitative differences were observed by comparing pTα mRNA expression patterns in T-ALLs with expression patterns known from thymic T-cell development. In contrast to thymocytes, maximum pTα a expression was found prior to the β-selection in immature T-ALLs [2,15]. Furthermore, pTα a was still expressed in mature T-ALLs, while pTα b was rarely detected. The variation of pTα a gene expression level throughout the immunophenotypical compartments is impressively higher among T-ALL subtypes (approximately 10.000-fold) than during intrathymic differentiation (approximately 80-fold) [15].
Even though pTα a protein expression has been rarely reported in thymocyte populations corresponding to immunophenotypical immature T-ALL cells, we clearly demonstrated pTα protein translation in immature T-ALL cells (Fig. 1D). Our finding is supported by Gounari et al. [11] who observed human CD25 protein expression in murine lymphoid restricted progenitors, controlled by the pTα promoter. Since pTα a expres- sion and function is physiologically down-regulated by pTα b in mature T-cells [2,15], it was surprising to observe pTα protein expression in mature T-ALLs. This finding correlates with unexpectedly persistent pTα a mRNA expression concomitant with lower than expected pTα b expression in mature T-ALLs (Fig. 1A, B) [15], suggesting an aberrantly maintained pTα signal due to a lack of physiological pTα down-regulation in mature T-ALLs. Importantly, the regulation of pTα mRNA-isoforms and pTα protein expression is unknown. Here, we interestingly observed a tendency of mRNA/protein proportion from high/low in immature T-ALLs inverting during further maturated phenotypes to low/high in mature T-ALLs (Fig. 1A, D). Whereas the known function of T-cell receptors is in general linked to defined stages, cellular environment and pTα/preTCR signal intensity, one might speculate that the mRNA/protein expression profile in the thymic window reflects the physiological differentiation; the expression profile in immature and mature T-ALL differs strikingly from this physiological situation, possibly presenting an abnormal and/or additional regulation of pTα gene/protein expression.
To better define the functional relevance of pTα in T-ALL cell lines, we used the src-PTK-I PP1 to block proximal pTα/preTCR signaling, which is mainly mediated through p56 Lck [22,23]. Initially, p56 Lck mRNA (Fig. 1C) and protein expression as well as tyrosinephosphorylation were analyzed (Fig. 2). The immature T-ALLs showed low levels of p56 Lck mRNA (Fig. 1C). On the protein level, HSB-2 showed strong p56 Lck and P-Tyr signals (Fig. 2C), whereas a second immature T-ALL cell line, RPMI-8402, exhibited lower p56 Lck and total phosphorylated protein levels. All thymic T-ALLs showed p56 Lck mRNA, protein expression and a corresponding P-Tyr signal. In the mature T-ALLs, strong p56 Lck mRNA and protein expression were detected with a lower corresponding P-Tyr signal (Fig. 2C).
Therefore, an IC 50 <10 µM PP1 was considered as specific inhibition of pTα/preTCR proximal signaling. Using these criteria, we observed functional pTα/preTCR expression in six out of eleven tested T-ALL cell lines (Fig. 3A).
The highest PP1 responsiveness (IC 50 : 0.96 µM) was observed in HSB-2 (Fig. 3A), one of two tested immature T-ALLs; further analyses could demonstrate that PP1 exposure resulted in apoptosis induction of HSB-2 (Figs 3B, 4A, B), clearly shown by nearly 90% annexin-V positive cells. In four out of six thymic T-ALL cell lines PP1 induced a G 1/0 arrest and caused moderate growth inhibition in one out of three mature T-ALL cell lines, without any evidence of apoptosis (Figs 3B, 4). All PP1 sensitive cell lines expressed the target protein p56 Lck and showed a corresponding P-Tyr signal (Fig. 2C). Two out of six thymic T-ALLs, which are early thymic T-ALLs according to the immunophenotype, did not show PP1 sensitivity, even though they expressed p56 Lck with corresponding P-Tyr signals (Fig. 2C). One might speculate, that in these PP1 non-sensitive thymic T-ALL cell lines alternative oncogenic pathways independent of phosphory-  lated p56 Lck deliver a more dominant survival advantage.
We were further interested in the high sensitivity of the immature T-ALL cell line HSB-2 to PP1, as pTα signaling in immature T-ALLs is very unlikely and suspected an oncogenic pathway other than pTα/preTCR. Interestingly, the chromosomal analysis of HSB-2 cells (data not shown) revealed the translocation t(1; 7)(q34; p34) [21,25], which causes independently of pTα a hyperactivation of p56 Lck (Fig. 2C). Even though the incidence of the translocation t(1; 7)(q34; p34) is considerably low at the initial diagnosis of a T-ALL, it has been speculated whether or not this translocation, or that Lck in general, is involved in later genetic events causing a transformation to a progressively more aggressive leukemia [14,21,25]. Furthermore, alterations of the chromosome 1p are frequently observed in human neoplastic disease. Approximately 10% of non-Hodgkin Lymphomas had genetic alteration at the site 1p31-36, potentially affecting Lck expression [14] and thereby represent an interesting therapeutical target for src-PTK-I as PP1.
The PP1-induced G 1/0 arrest in thymic T-ALL cell lines supports the hypothesis that pTα/preTCR sustains proliferation in the leukemic thymic compartment, in which pTα activation is physiologically relevant for G 1/0 to S-phase transition and the arrested leukemogenic clone might be supported from this potent survival mediator [5,9,23]. Interestingly, not all thymic T-ALLs respond in the defined PP1 concentration range (<10 µM) arguing for a very tightly defined time window of malignant arrest in which pTα is relevant: PP1 unresponsive cell lines are immunophenotypically early thymic T-ALLs, whereas the responsive cell lines are considered to be in the intermediate and late thymic developmental time window. With respect to T-cell leukemogenesis further dissection of the Notch/pTα/CyclinD3 signaling axis is needed, as Notch1 or CyclinD3 inhibition, upstream or downstream of the pTα/preTCR, also cause G 1/0 arrest predominantly in thymic T-ALLs [8,18,24].
In summary, our data point at the crucial role of pTα in defined subtypes of T-ALL mediating survival advantages for the malignant cell-type. Additionally, using the src-PTK-I PP1, we demonstrated that p56 Lck also independently of pTα represents a promising therapeutical target in neoplastic T-cell diseases.