7 research outputs found
Induction of Cell Membrane Protrusions by the N-terminal Glutaredoxin Domain of a Rare Splice Variant of Human Thioredoxin Reductase 1
Get PDF18 páginas, 5 figuras, 1 esquema, 1 pelĂcula.The human thioredoxin system has a wide range of functions in cells including regulation of cell proliferation and differentiation, immune system modulation, antioxidant defense, redox control of transcription factor activity, and promotion of cancer development. A key component of this enzymatic system is the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene. Transcription of TXNRD1 involves alternative splicing, leading to a number of transcripts also encoding isoforms of TrxR1 that differ from each other at their N-terminal domains. Here we have studied the TXNRD1_v3 isoform containing an atypical N-terminal glutaredoxin (Grx) domain. Expression of the transcript of this isoform was found predominantly in testis but was also detected in ovary, spleen, heart, liver, kidney, and pancreas. By immunohistochemical analysis in human testis with antibodies specific for the Grx domain of TXNRD1_v3, the protein was found to be predominantly expressed in the Leydig cells. Expression of the TXNRD1_v3 transcript was also found in several cancer cell lines (HCC1937, H23, A549, U1810, or H157), and in HeLa cells, it was induced by estradiol or testosterone treatments. Surprisingly, green fluorescent protein fusions with the complete TXNRD1_v3 protein or with only its Grx domain localized to distinct cellular sites in proximity to actin, and furthermore, had a potent capacity to rapidly induce cell membrane protrusions. Analyses of these structures suggested that the Grx domain of TXNRD1_v3 localizes first in the emerging protrusion and is then followed into the protrusions by actin and subsequently by tubulin. The results presented thus reveal that TXNRD1_v3 has a unique and distinct expression pattern in human cells and suggest that the protein can guide actin polymerization in relation to cell membrane restructuring.This study was supported by grants from the Swedish Cancer Society, the Swedish Research Council (Medicine), the Ă…ke Wibergs Foundation and Karolinska Institutet.Peer reviewe
Molecular and cellular strategies to enhance efficacy of T cell-based cancer therapy
Full text linkA combined approach of vaccination with β2-microglobulin (β2m)-deficient
dendritic cells (DCs) and granulocyte-macrophage colony-stimulating
factor (GM-CSF) as a potent adjuvant may link cellular and molecular
strategies to further enhance antitumor T-cell responses. T lymphocytes
can mediate a potent antitumor immune response. CD8+ T lymphocytes screen
and recognize antigen in complex with a major histocompatibility complex
(MHC) class I heavy chain (HQ and the β2m light chain. Metastatic cells
commonly escape from "conventional" T lymphocyte-mediated recognition and
elimination as a result of impaired cell surface expression of MHC class
I antigen molecules. This impaired cell surface expression can be caused
by loss or down-regulation of expression of different components of the
MHC class I antigen processing machinery, such as MHC class I HCs, β2m,
or the transporter associated with antigen processing (TAP). Such immune
evasion poses a problem for autologous T cell-based cancer therapy.
In the first study we demonstrated protection against growth Of
β2m-deficient tumor cells in syngeneic C57Bl/6 (B6) mice, following
vaccination with β2m-deficient DCs. In vitro analysis of an effector cell
population from vaccinated mice pointed to that CD3+ cells had been
generated with the capability to induce apoptosis in syngeneic
β2m-deficient tumor and nonmalignant cells. Further investigation of
target cell recognition suggested that also tumor target cells lacking
expression of classical MHC class I HCs and functional TAP were
recognized by CD3+ effector cells from vaccinated mice. This study points
to a new possible strategy to counteract the growth of metastatic cells.
The cytokine composition in the tumor microenvironment is a critical
factor for an effective antitumor immune response. GM-CSF has been shown
to be a very promising cytokine in antitumor immunomodulation.
Continuously high concentrations of GM-CSF in the local tumor environment
seem to be crucial to reach a therapeutic threshold. Such a favorable
cytokine milieu can promote recruitment of DCs and augment DC activation
with increased number of DCs expressing MHC and co-stimulatory molecules.
GM-CSF can enhance tumor infiltration of T lymphocytes and their
cross-priming. Furthermore, GM-CSF seems to trigger an increased and
significantly more effective tumor lysis mediated by lymphocytes. GM-CSF
has elicited antitumor immune responses in animal studies and clinical
trials. However, the clinical efficacy has been limited, with local
GM-CSF levels being therapeutically insufficient or systemic toxicity
being a limiting factor.
In the second study, we developed and characterized a novel GM-CSF
expression vector, pAD-HotAmp-GM-CSF, which can provide heat-inducible
high-level expression of GM-CSF. In cytokine immunotherapy of cancer it
is critical to deliver sufficiently high local cytokine concentrations in
order to reach the therapeutic threshold needed for clinical efficacy.
The novel vector, pAD-HotAmp-GM-CSF successfully integrates inducible and
amplifying elements into a one-plasmid system. Moderate hyperthermia at
42°C for 30 min induced amplification of GM-CSF expression in
pAD-HotAmp-GM-CSF that was over 2,8 fold higher than levels achieved with
the prototypical human cytomegalovirus (CMV) promoter. Thus, the
inducible amplifier vector, pAD-HotAmp-GM-CSF, represents a novel system
for regulated and enhanced GM-CSF expression, which enables both greater
efficacy and safety in cytokine immunotherapy of cancer
Cancer vaccine strategies and studies of human thioredoxin reductase splice variants
Get PDFCancer involves abnormal, uncontrolled proliferation of cells, and
evasion from the immune system. Immune evasion can be caused by defects
in one or more of the components of the major histocompatibility complex
class I antigen machinery, e.g. the beta2-microglobulin (beta2m)
molecule. Here, it was demonstrated that vaccination of mice with
beta2m-deficient dendritic cells, prior to a challenge with a tumorigenic
dose of beta2mfree tumor cells of syngeneic origin, protected the
vaccinated mice from tumor development. Antitumor immune reactions depend
upon the cytokine composition in the tumor microenvironment. An efficient
antitumor immune response correlates with local high-level expression of
the granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine. A
heat-inducible DNA vector for amplified GM-CSF expression was herein
constructed, which showed almost three-fold greater expression levels
upon induction by hyperthermia than a prototypic CMV promoter construct,
while both constructs had similar basal levels.
Thioredoxin reductase (TrxR) is an essential redox-active selenoenzyme
important for human health and disease. For example, TrxR is involved in
many steps of tumorigenesis, with TrxR expression often upregulated in
tumors. Moreover, TrxR is known to be a target of platinum-based drugs,
which are used in anticancer therapy. Data presented here show that in
organotypic cochlear cultures, direct exposure of equimolar
concentrations of the frequently ototoxic platinum-based drug cisplatin
and the rarely ototoxic oxaliplatin both targeted cochlear TrxR and
caused hair loss to similar degrees. These findings emphasize the
importance of understanding pharmacodynamics (oxaliplatin does not reach
the inner ear in vivo) and may add to improve therapeutic strategies in
order to lower patient toxicity caused by platinumbased drugs.
TrxR1 is the most abundant TrxR isoenzyme, which is predominantly found
in the cytosol. The gene encoding TrxR1 TXNRD1 harbors a complex
genomic structure, leading to numerous splice variants. Peculiarly, the
v3 splice variant encompasses a unique glutaredoxin domain, transcription
of which is guided by an alternative promoter, which is located upstream
of the TrxR1 core promoter. Expression of v3 was herein found in the
developing human cochlea and the Leydig cells of the testis. Transcripts
encoding v3 were detected in human heart, liver, spleen, ovary, kidney
and pancreas, as well as several cancer cell lines. Several cell
stressors (including starvation, hypoxia, etoposide, rapamycin,
nocodazole) increased v3 promoter activity and v3 expression, while
simultaneously repressing the TrxR1 core promoter activity and expression
of the classical form of TrxR1. Translation of v3 was found to be
IRESdependent.´In particular, prolonged starvation induced expression of
v3, dynamic formation of membrane protrusions to which endogenous v3
was localized and an increase in cell motility, all of which correlated
in time. Recombinant overexpression of v3 in transfected cell lines
induced a similar phenotype, with the dynamic formation of membrane
protrusions. The data presented herein indicate that the formation of
membrane protrusions and increased cell motility are linked with each
other and with the induction of v3 expression.
In conclusion, cell-based and DNA vector-based cancer vaccine strategies
were studied with the potential to be used in therapeutic cancer vaccine
approaches. The splice variant v3 may possibly be targeted in anticancer
therapy to interfere with cancer cell motility and ultimately thus, the
formation of metastases
Induction of Cell Membrane Protrusions by the N-terminal Glutaredoxin Domain of a Rare Splice Variant of Human Thioredoxin Reductase 1
Get PDF18 páginas, 5 figuras, 1 esquema, 1 pelĂcula.The human thioredoxin system has a wide range of functions in cells including regulation of cell proliferation and differentiation, immune system modulation, antioxidant defense, redox control of transcription factor activity, and promotion of cancer development. A key component of this enzymatic system is the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene. Transcription of TXNRD1 involves alternative splicing, leading to a number of transcripts also encoding isoforms of TrxR1 that differ from each other at their N-terminal domains. Here we have studied the TXNRD1_v3 isoform containing an atypical N-terminal glutaredoxin (Grx) domain. Expression of the transcript of this isoform was found predominantly in testis but was also detected in ovary, spleen, heart, liver, kidney, and pancreas. By immunohistochemical analysis in human testis with antibodies specific for the Grx domain of TXNRD1_v3, the protein was found to be predominantly expressed in the Leydig cells. Expression of the TXNRD1_v3 transcript was also found in several cancer cell lines (HCC1937, H23, A549, U1810, or H157), and in HeLa cells, it was induced by estradiol or testosterone treatments. Surprisingly, green fluorescent protein fusions with the complete TXNRD1_v3 protein or with only its Grx domain localized to distinct cellular sites in proximity to actin, and furthermore, had a potent capacity to rapidly induce cell membrane protrusions. Analyses of these structures suggested that the Grx domain of TXNRD1_v3 localizes first in the emerging protrusion and is then followed into the protrusions by actin and subsequently by tubulin. The results presented thus reveal that TXNRD1_v3 has a unique and distinct expression pattern in human cells and suggest that the protein can guide actin polymerization in relation to cell membrane restructuring.This study was supported by grants from the Swedish Cancer Society, the Swedish Research Council (Medicine), the Ă…ke Wibergs Foundation and Karolinska Institutet.Peer reviewe
