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Transforming growth factor-β (TGF-β) potently induces apoptosis in Burkitt's lymphoma (BL) cell lines and in explanted primary human B lymphocytes. The physiological relevance and mechanism of TGF-β-mediated apoptosis induction in these cells remains to be determined. Here we demonstrate the requirement for TGF-β-mediated regulation of BIK and BCL-XL to activate an intrinsic apoptotic pathway in centroblastic BL cells. TGF-β directly induced transcription of BIK and a consensus Smad-binding element identified in the BIK promoter recruits TGF-β-activated Smad transcription factor complexes in vivo. TGF-β also transcriptionally repressed expression of the apoptosis inhibitor BCL-XL. Inhibition of BCL-XL sensitised BL cells to TGF-β-induced apoptosis whereas overexpression of BCL-XL or suppression of BIK by shRNA, diminished TGF-β-induced apoptosis. BIK and BCL-XL were also identified as TGF-β target genes in purified normal human centroblast B cells and immunohistochemical analyses of tonsil tissue revealed widespread TGF-β receptor-regulated Smad activation and a focal pattern of BIK expression. Furthermore, using a selective inhibitor of the TGF-β receptor we provide evidence that autocrine TGF-β signalling through ALK5 contributes to the default apoptotic programme in normal human centroblasts undergoing spontaneous apoptosis. Our data suggests that TGF-β may act as a physiological mediator of human germinal centre homoeostasis by regulation of BIK and BCL-XL.Cell Death and Differentiation (2009) 16, 593–602; doi:10.1038/cdd.2008.183; published online 9 January 2009 [ABSTRACT FROM AUTHOR]

Transforming growth factor β-1 (TGF-β) acts as both a tumour suppressor and a tumour promoter in a context-dependent manner. The tumour-promoting activities of TGF-β are likely to result from a combination of Smad and non-Smad signalling pathways but remain poorly understood. Here we show that TGF-β-mediated activation of RhoA is dependent on the kinase activity of ALK5 and that continuous ALK5 activity maintains basal RhoA–ROCK signalling, cell morphology and actin dynamics in serum-starved rodent fibroblasts independently of Smad2, Smad3 and Smad4. In immortalized human diploid fibroblasts, we show that oncogenic rewiring by transduction of V12HaRas instigates regulation of RhoA–ROCK signalling through an autocrine TGF-β1–ALK5 pathway. Furthermore, we show that ALK5-mediated activation of RhoA is required for efficient V12HaRas, V-Raf and V600EBRAF transformation and V12HaRas-mediated anchorage-independent growth. These findings identify a new pro-oncogenic activity of TGF-β and indicate that tumours harbouring V12HaRas and V600EBRAF mutations may be susceptible to TGF-β signalling inhibitors.Oncogene (2009) 28, 983–993; doi:10.1038/onc.2008.449; published online 15 December 2008 [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) potently induces apoptosis in Burkitt's lymphoma (BL) cell lines and in explanted primary human B lymphocytes. The physiological relevance and mechanism of TGF-β-mediated apoptosis induction in these cells remains to be determined. Here we demonstrate the requirement for TGF-β-mediated regulation of BIK and BCL-XL to activate an intrinsic apoptotic pathway in centroblastic BL cells. TGF-β directly induced transcription of BIK and a consensus Smad-binding element identified in the BIK promoter recruits TGF-β-activated Smad transcription factor complexes in vivo. TGF-β also transcriptionally repressed expression of the apoptosis inhibitor BCL-XL. Inhibition of BCL-XL sensitised BL cells to TGF-β-induced apoptosis whereas overexpression of BCL-XL or suppression of BIK by shRNA, diminished TGF-β-induced apoptosis. BIK and BCL-XL were also identified as TGF-β target genes in purified normal human centroblast B cells and immunohistochemical analyses of tonsil tissue revealed widespread TGF-β receptor-regulated Smad activation and a focal pattern of BIK expression. Furthermore, using a selective inhibitor of the TGF-β receptor we provide evidence that autocrine TGF-β signalling through ALK5 contributes to the default apoptotic programme in normal human centroblasts undergoing spontaneous apoptosis. Our data suggests that TGF-β may act as a physiological mediator of human germinal centre homoeostasis by regulation of BIK and BCL-XL.Cell Death and Differentiation (2009) 16, 593–602; doi:10.1038/cdd.2008.183; published online 9 January 2009 [ABSTRACT FROM AUTHOR]

Background: For patients with early American Joint Committee on Cancer (AJCC)-stage melanoma the combined loss of the autophagy regulatory protein AMBRA1 and the terminal differentiation marker loricrin in the peritumoral epidermis is associated with a significantly increased risk of metastasis., Objectives: The aim of the present study was to evaluate the potential contribution of melanoma paracrine transforming growth factor (TGF)-β signalling to the loss of AMBRA1 in the epidermis overlying the primary tumour and disruption of epidermal integrity., Methods: Immunohistochemistry was used to analyse AMBRA1 and TGF-β2 in a cohort of 109 AJCC all-stage melanomas, and TGF-β2 and claudin-1 in a cohort of 30 or 42 AJCC stage I melanomas, respectively, with known AMBRA1 and loricrin (AMLo) expression. Evidence of pre-ulceration was analysed in a cohort of 42 melanomas, with TGF-β2 signalling evaluated in primary keratinocytes., Results: Increased tumoral TGF-β2 was significantly associated with loss of peritumoral AMBRA1 (P < 0·05), ulceration (P < 0·001), AMLo high-risk status (P < 0·05) and metastasis (P < 0·01). TGF-β2 treatment of keratinocytes resulted in downregulation of AMBRA1, loricrin and claudin-1, while knockdown of AMBRA1 was associated with decreased expression of claudin-1 and increased proliferation of keratinocytes (P < 0·05). Importantly, we show loss of AMBRA1 in the peritumoral epidermis was associated with decreased claudin-1 expression (P < 0·05), parakeratosis (P < 0·01) and cleft formation in the dermoepidermal junction (P < 0·05)., Conclusions: Collectively, these data suggest a paracrine mechanism whereby TGF-β2 causes loss of AMBRA1 overlying high-risk AJCC early-stage melanomas and reduced epidermal integrity, thereby facilitating erosion of the epidermis and tumour ulceration., (© 2021 The Authors. British Journal of Dermatology published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.)

Background: Recessive dystrophic epidermolysis bullosa (RDEB) is associated with a high mortality rate due to the development of life-threatening, metastatic cutaneous squamous cell carcinoma (cSCC). Elevated transforming growth factor-beta (TGF-β) signalling is implicated in cSCC development and progression in patients with RDEB., Objectives: To determine the effect of exogenous and endogenous TGF-β signalling in RDEB cSCC with a view to assessing the potential of targeting TGF-β signalling for RDEB cSCC therapy., Methods: A panel of 11 patient-derived RDEB cSCC primary tumour keratinocyte cell lines (SCCRDEBs) were tested for their signalling and proliferation responses to exogenous TGF-β. Their responses to TGF-β receptor type-1 (TGFBR1) kinase inhibitors [SB-431542 and AZ12601011 (AZA01)] were tested using in vitro proliferation, clonogenicity, migration and three-dimensional invasion assays, and in vivo tumour xenograft assays., Results: All SCCRDEBs responded to exogenous TGF-β by activation of canonical SMAD signalling and proliferative arrest. Blocking endogenous signalling by treatment with SB-431542 and AZ12601011 significantly inhibited proliferation (seven of 11), clonogenicity (six of 11), migration (eight of 11) and invasion (six of 11) of SCCRDEBs. However, these TGFBR1 kinase inhibitors also promoted proliferation and clonogenicity in two of 11 SCCRDEB cell lines. Pretreatment of in vitro TGFBR1-addicted SCCRDEB70 cells with SB-431542 enhanced overall survival and reduced tumour volume in subcutaneous xenografts but had no effect on nonaddicted SCCRDEB2 cells in these assays., Conclusions: Targeting TGFBR1 kinase activity may have therapeutic benefit in the majority of RDEB cSCCs. However, the potential tumour suppressive role of TGF-β signalling in a subset of RDEB cSCCs necessitates biomarker identification to enable patient stratification before clinical intervention., (© 2020 British Association of Dermatologists.)

Expression of the lytic cycle genes of Epstain-Barr virus (EBV) is induced in type I Burkitt's lymphoma-derived cells by treatment with phorbol esters (e.g., phorbol myristate acetate [PMA]), anti-immunoglobulin, or the cytokine transforming growth factor beta (TGF-beta). Concomitantly, all these agents induce apoptosis as judged by a sub-G1 fluorescence-activated cell sorter (FACS) profile, proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. However, caspase activation is not required for induction of the lytic cycle since the latter is not blocked by the caspase inhibitor ZVAD. Furthermore, not all agents that induce apoptosis in these cultures (for example, cisplatin and ceramide) induce the EBV lytic programme. Although it is closely associated with the lytic cycle, apoptosis is neither necessary nor sufficient for its activation. Multiparameter FACS analysis of cultures treated with PMA, anti-Ig, or TGF-beta revealed BZLF1-expressing cells distributed in different phases of the cell cycle according to which inducer was used. However, BZLF1-positive cells did not appear to undergo apoptosis and accumulate with a sub-G1 DNA content, irrespective of the inducer used. This result, which suggests that lytic gene expression is protective, was confirmed and extended by immunofluorescence staining doubled with TUNEL analysis. BZLF1- and also gp350-expressing cells were almost always shown to be negative for TUNEL staining. Similar experiments using EBV-positive and -negative subclones of Akata BL cells carrying an episomal BZLF1 reporter plasmid confirmed that protection from apoptosis was associated with the presence of the EBV genome. Finally, treatment with phosphonoacetic acid or acyclovir prior to induction with PMA, anti-Ig, or TGF-beta blocked the protective effect in Mutu-I cells. These data suggest that a late gene product(s) may be particularly important for protection against caspase activity and cell death.

TGF-beta is a potent inducer of apoptosis in many Burkitt's lymphoma (BL) cell lines. In this study, we characterize this apoptotic process in the EBV-negative BL41 cell line. Induction of apoptosis was detected as early as 8 h after TGF-beta treatment, as assayed by TUNEL and poly(ADP-ribose) polymerase cleavage. FACS analysis demonstrates that this proceeds predominately from the G1, but also from the G2/M phases of the cell cycle. We observed no early detectable changes in the steady-state levels of Bcl-2 and several of its family members after TGF-beta treatment. We detected cleavage of caspases 2, 3, 7, 8, and 9 into their active subunits. Consistent with the involvement of these enzymes in TGF-beta-mediated apoptosis, the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(Ome)-flouromethylketone (ZVAD-fmk) blocked TGF-beta-induced apoptosis and revealed a G1 arrest in treated cells. Use of specific caspase inhibitors revealed that the induction of apoptosis is caspase 8 dependent, but caspase 3 independent. Activation of caspase 8 has been shown to be a critical event in death receptor-mediated apoptosis. However, TGF-beta treatment of BL41 cells was found not to affect the cell surface expression of Fas, TNF-R1, DR3, DR4, or DR5, or the steady-state expression levels of Fas ligand, TNF-R1, DR3, DR4, and DR5. Furthermore, blocking experiments indicated that TGF-beta-mediated apoptosis is not dependent on Fas ligand, TNF-alpha, tumor necrosis-like apoptosis-inducing ligand, or TNF-like weak inducer of apoptosis signaling. Therefore, it appears that TGF-beta induces apoptosis in BL cell lines via caspase 8 in a death receptor-independent fashion.

The pleiotropic cytokine TGF-beta1 is a member of a large family of related factors involved in controlling cell proliferation, differentiation and apoptosis. TGF-beta ligands interact with a complex of type I and type II transmembrane serine/threonine kinases and they transmit their signals to the nucleus via a family of Smad proteins. A panel of over 20 Burkitt's lymphoma (BL) cell lines has been compiled including those that are Epstein-Barr virus (EBV) negative, those that carry EBV with a restricted pattern of EBV latent gene expression (group I) and those that express the full range of latent EBV genes (group III), together with selected EBV-transformed lymphoblastoid cell lines (LCLs). Most of the EBV-negative and group I BL cell lines underwent apoptosis or a G(1) arrest in response to TGF-beta1 treatment. In contrast, group III cell lines and LCLs were completely refractory to these effects of TGF-beta1. All of the cell lines expressed the TGF-beta pathway Smads and the TGF-beta type I receptor. Lack of responsiveness to TGF-beta1 appears to correlate with a down-regulation of TGF-beta type II receptor expression. Studies of EBV-converted and stably transfected BL cell lines demonstrated that the EBV gene LMP-1 is neither necessary nor sufficient to block the TGF-beta1 response.

In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.