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Migration of human glioma cells (hGCs) within the brain parenchyma makes glioblastoma one of the most aggressive and lethal tumors. Studies of the cellular and molecular mechanisms underlying hGC migration are hindered by the limitations of existing glioma models. Here we developed a dorsal root ganglion axon-oligodendrocyte-hGC co-culture to study in real time the migration and interaction of hGCs with their microenvironment. hGCs interact with myelinated and non-myelinated axons through the formation of pseudopodia. Isolation of pseudopodia-localized polysome-bound RNA reveals transcripts of Lck, Paxillin, Crk-II, and Rac1 that undergo local translation. Inhibition of Lck phosphorylation using a small-molecule inhibitor (Lck-I), blocks the phosphorylation of Paxillin and Crk-II, the formation of pseudopodia and the migration of hGCs. In vivo intraventricular administration of the Lck-I using an orthotopic xenograft glioma model, results in statistically significant inhibition of tumor size and significant down-regulation of Nanog-targeted genes, which are associated with glioblastoma patient survival. Moreover, treatment of human glioma stem cells (hGSCs) with Lck-I, results in significant inhibition of self-renewal and tumor-sphere formation. The involvement of Lck in different levels of glioma malignant progression, such as migration, tumor growth, and regulation of cancer stemness, makes Lck a potentially important therapeutic target for human glioblastomas.

Tyrosine kinases of the Src family have been implicated in key biological processes. Here, we provide evidence that p56(Lck), a lymphoid-specific Src kinase, is involved in the activation of the mitochondrial apoptosis pathway. Lck-deficient T cells were completely resistant to anticancer drugs. In contrast, apoptosis sensitivity to death receptors was not altered, indicating a specific interference of Lck with the mitochondrial pathway. Re-expression of Lck restored sensitivity to drug-induced apoptosis and triggered mitochondrial cytochrome c release and caspase activation. Further analysis identified that the sensitization by Lck was independent of classical mediators of T-cell signaling, but essentially involved the Bcl-2 protein Bak. Expression of Bak was completely absent in Lck-deficient cells, while re-expression of Lck transcriptionally triggered Bak expression and conferred sensitivity to apoptosis, associated with a proapoptotic conformational change of Bak. Furthermore, in vitro the truncated fragment of Bid specifically activated Bak and cytochrome c release only from mitochondria of Lck-expressing cells. These results do not only demonstrate a sentinel role of Lck in drug resistance but also delineate a hitherto unknown pathway of Src kinases in regulation of Bcl-2 proteins.

The induction of apoptosis requires the activation of a highly coordinated signaling network ultimately leading to the activation of caspases. In previous experiments we and others have shown that the tyrosine kinase Lck is required for adequate apoptosis induction in response to ionizing radiation, ceramide incubation and overexpression of the HIV-TAT protein. However, the position of Lck within given apoptotic signaling cascades remains unclear. We therefore aimed to define the role of Lck during radiation-induced apoptosis. Apoptosis induction in response to ionizing radiation, CD95 or TRAIL receptor stimulation was determined in Jurkat T-cells, the Lck-deficient Jurkat clone JCaM1.6- and Lck-retransfected JCaM1.6/Lck. No apoptosis, release of cytochrome c, breakdown of the mitochondrial potential were detectable during the first 48 h after irradiation of JCaM1.6 cells. In parallel, no activation of caspase-9, -8 and -3 was detectable. Since mitochondrial apoptosis pathways act within a feedback mechanism during death-receptor-mediated apoptosis, the influence of the Lck defect on CD95/Fas/Apo-1-L or TRAIL-induced apoptosis was also tested. Both stimuli induced apoptosis in Lck-deficient cells. However, the kinetics of apoptosis induction determined by caspase-8, -9 and -3 activation as well as deltapsi(m) breakdown was slowed. We conclude that the Lck deficiency influences early steps during radiation-induced mitochondrial alterations.

The activities of Src-family non-receptor tyrosine kinases are regulated by structural changes that alter the orientation of key residues within the catalytic domain. In this study, we investigate the effects of activation loop mutations on regulation of the lymphocyte-specific kinase Lck (p56lck). Substitution of 5 - 7 residues amino terminal to the conserved activation loop tyrosine (Y394) increases kinase activity and oncogenic potential regardless of regulatory C-terminal tail phosphorylation levels (Y505), while most mutations in the 13 residues carboxyl to Y394 decrease kinase activity. Phosphorylation of the C-terminal regulatory tail is carried out by the cytosolic tyrosine kinase Csk and we find that mutations upstream or downstream of Y394 or mutation of Y394 do not affect the level of Y505 phosphorylation. In addition, we report that mutations on either side of Y394 affect substrate specificity in vivo. We conclude that the high degree of conservation across the entire activation loop of Src-family kinases is critical for normal regulation of kinase activity and oncogenicity as well as substrate selection. Oncogene (2000) 19, 3961 - 3961.

Induction of apoptosis is a hallmark of cytostatic drug and radiation-induced cell death in human lymphocytes and lymphoma cells. However, the mechanisms leading to apoptosis are not well understood. We provide evidence that ionizing radiation induces a rapid activation of caspase-8 (FLICE) followed by apoptosis independently of CD95 ligand/receptor interaction. The radiation induced cleavage pattern of procaspase-8 into mature caspase-8 resembled that following CD95 crosslinking and resulted in cleavage of the proapoptotic substrate BID. Overexpression of dominant-negative caspase-8 interfered with radiation-induced apoptosis. Caspase-8 activation by ionizing radiation was not observed in cells genetically defective for the Src-like tyrosine kinase Lck. Cells lacking Lck also displayed a marked resistance towards apoptosis induction upon ionizing radiation. After retransfection of Lck, caspase-8 activation and the capability to undergo apoptosis in response to ionizing radiation was restored. We conclude that radiation activates caspase-8 via an Lck-controlled pathway independently of CD95 ligand expression. This is a novel signaling event required for radiation induced apoptosis in T lymphoma cells.

The failure of signal transduction in the JCaM1 cell line was associated with the presence of an abnormal lck mRNA deleted of the exon 7 encoding for an inactive p56lck kinase. Our study of the lck mRNA from various T cell lines and from peripheral blood lymphocytes of healthy donors has revealed the presence of both complete and exon 7-deleted lck transcripts. Thus the exon 7-deleted lck transcript initially described in the JCaM1 mutant cell line, arises from an alternative splicing event occurring in each cells expressing the lck gene. Genomic DNA sequencing of the lck exons 6-8 portion from both the mutant JCaM1 and its parental Jurkat cell lines revealed as the only difference, the presence of a A to G mutation within the 5' splice site of intron 7 in the JCaM1 cell line DNA. To demonstrate the role of this point mutation in the lck pre-mRNA maturation, COS cells were transfected by lck minigenes from the Jurkat and JCaM1 cell lines. In COS cells transfected with minigene from the Jurkat cell line both lck transcripts (with and without exon 7) were observed whereas only the exon 7-spliced lck transcript was observed in COS cells transfected with minigene from the JCaM1 cell line. Thus the mutation is per se responsible for the deletion of exon 7 and the absence of complete lck mRNA in the JCaM1 cell line. Presence of a restriction site (HphI) in the 5' splice site of lck intron 7 from Jurkat DNA allowed to confirm the presence of the mutation on both alleles in the JCaM1 cell line.

The gfi-1 gene encodes a zinc finger containing protein that is specifically expressed in T-lymphocytes and is a frequent target of proviral insertion in T-cell lymphoma provoked by infection with MoMuLV--a non acute transforming retrovirus. Expression of a gfi-1 transgene targeted to T-cells by the lck proximal promoter provokes a reduction of peripheral CD4 and CD8 positive T-cells but nevertheless weakly predisposes transgenic animals for the development of T-cell lymphoma. Forced coexpression of the serine/threonine kinase Pim-1 can partially restore normal T-cell numbers in double pim-1/gfi-1 transgenic mice. Moreover, the combinatorial expression of Pim-1 and Gfi-1 leads to accelerated development of T-cell lymphoma with a mean latency period of 114 days. A similar accelerated rate of lymphoma development was observed when lck-gfi-1 mice were crossed with mice that carry a L-myc gene targeted to be expressed at high levels in T-cells. The results show that gfi-1 can act with low activity as a dominant oncogene when overexpressed but also demonstrate that it is most efficient only in the presence of a cooperative partner protein as for example Pim-1 or L-Myc. In addition, the results suggest that Pim-1 and Gfi-1 are acting synergistically in both T-cell lymphomagenesis and T-cell development.

The product of the proto-oncogene p56lck is a non-receptor tyrosine kinase member of the Src family. It is found in T cells (Marth et al., 1985, 1988) and in the mouse brain (Omri et al., 1996; Van Tan et al., 1996). In this report, we describe experiments showing that Lck is present in the mouse retina neurons. Lck gene expression was identified after isolating and sequencing the specific 5' and 3' part of the cDNA obtained by RT-PCR. In adult retina Lck immunoreactivity was most abundant in photoreceptor cells and within the outer plexiform layers. Staining was also observed in the inner nuclear and plexiform layers. In transgenic mice, the disruption of the Lck gene had serious consequences on the organization of the retina causing retinal dysplasia. These mice have partial retinal detachment with infolding and rosette formation in the photoreceptor sheet. These retinal abnormalities observed in Lck deficient mice lead to the loss of normal architecture of the photoreceptor and the inner nuclear layers, and provide an important role of Lck protein in the retina development. The lack of the Lck protein produces a spectrum of retinal pathology that resembles human retinopathy of prematurity (ROP).

The lymphoid-specific protein tyrosine kinase, p56lck which is essential for both T cell development and function, is aberrantly expressed in colon and small lung carcinoma lines. In this paper, we demonstrate p56lck is also expressed in colon tumour biopsies due predominantly or exclusively to the use of the lck type I promoter. In T leukaemia lines, the lck type I promoter requires binding sites for both Ets- and Myb-related transcription factors. In contrast, in colon tumour lines the activation of the lck type I promoter requires the Ets but not the Myb binding site. In these lines, a consensus binding site for HMG-related transcription factors, AACAAAG, is required for efficient lck type I promoter activity. Sox-4 is a candidate transcription factor for binding and activating the lck type l promoter in colon carcinoma cells. Co-expression of Ets-1 and Sox-4, but neither protein alone, was sufficient to activate the lck type l promoter in HeLa cells which do not normally express lck transcripts. These results suggest that aberrant expression of p56lck from the lck type l promoter in colon carcinoma arises from transcriptional activation mediated by Ets- and HMG-related transcription factors.