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,1
,
* Oncogenetic and
Hematological Laboratories,
Department of Internal Medicine, Meir Medical Center,
Kfar Saba and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
1Correspondence: Oncogenetic Laboratory, Meir Medical Center, Kfar Saba 44281, Israel. E-mail: druckerl{at}clalit.org.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: CD82 • CD81 • CAG • RPMI 8226
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The initial adhesion of the cells affect resistance to apoptosis and augmentation of cytokines and growth factors secretion, which mediate both growth and survival of MM cells (2
, 3)
. Moreover, subsequent changes in the adhesion molecule profile are associated with tumor cell migration into peripheral blood during progressive disease (2)
. Targeting the myeloma cell in its bone marrow microenvironment is perceived as fundamental for effective treatment of this disease.
In this study we postulated that to the initial orchestration of exterior–interior cell signaling, so crucial for MM cells, might contribute a family of ubiquitously expressed cell membrane proteins called tetraspanins (4
5
6
7)
. Tetraspanins modulate a variety of fundamental biological processes such as adhesion, migration, proliferation, and fusion by functioning as organizers of multimolecular membrane complexes, termed "tetraspanin-enriched microdomains" (TEMs) (4
5
6
7)
. Tetraspanins are often deregulated in malignant diseases. An association between tetraspanin expression and tumor progression has been observed (CD9 and CD231 in leukemia; CO-029 and SAS in carcinomas and sarcomas, respectively; CD63 in melanoma) (5
, 7)
. Moreover, the expression of CD9 and/or CD82 has also been reported to inversely correlate with metastatic potential of various cancers (8)
. Both positive (CD9 in astrocyte tumors and gastric cancer; NET-1 in cervical cancer) and negative (CD9 in melanoma and lung cancers; CD82 in prostate carcinoma) correlations between tetraspanin expression and tumor progression have been reported (5)
. The correlation of tetraspanin expression with prognosis is incongruous; for example, increased CD151 expression is indicative of poor prognosis in colon and lung cancer in contrast to CD9/CD82 expression, which indicates a good prognosis (7)
. Thus, the relationship of tetraspanin expression and cancer varies with members of the tetraspanin superfamily and in tumors of different origin. A role for tetraspanins in tumor biology has also been confirmed by transfection studies (7)
. Their involvement in attenuation of growth factor-induced signaling in cancer and regulation of metalloproteinase function was reported. Despite accumulating data that underscore the functional importance and increasing prominence of the tetraspanins, most family members do not carry out typical cell-surface receptor-ligand binding functions and the full extent of their modulation remains unclear (4
, 5)
.
We have previously established the down-regulation of tetraspanins in myeloma cell lines (9)
. Specifically, CD82 was absent in all five assayed MM cell lines (RPMI 8226, U266, ARP1, ARK, CAG), and CD81 was absent in 4 of the lines and minimally expressed in RPMI 8226 (9)
. CD81 and CD82 down-regulation was also determined in malignant plasma cells of myeloma patients’ bone marrow samples (9)
. This reduced expression is especially prominent in comparison to their widespread distribution in peripheral blood leukocytes (10)
. Semiquantitative multiplex RT-polymerase chain reaction (RT-PCR) employed for analysis of tetraspanin mRNA baseline expression depicted reduced levels of all tetraspanin transcripts and absence of at least one in all MM cell lines (9)
.
Thus, we aimed to determine the role and significance of the tetraspanin re-expression on myeloma cell viability, survival, adhesion, migration, and invasion. In this study we introduced into MM cells plasmid vectors coding fusion proteins of the tetraspanins and enhanced green fluorescence protein (eGFP). Our results show that the tetraspanins have differential effects depending on specific vector orientation and its protein fusion. Vectors with a free tetraspanin N terminus induced myeloma cell death, whereas constructs with a free C-terminus reduced adhesion, migration, and invasion of the transfected MM cell lines.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cell lines PC3 (human prostate cancer) and Jurkat (human T cell leukemia), kindly provided by the Collgard Company (Petach Tikva, Israel), were cultured in RPMI 1640 supplemented with 10% FBS. Jurkat cells were also supplemented with sodium pyruvate, HEPES buffer, and nonessentials amino acids (all were purchased from Biological Industries, Beit Haemek, Israel).
Construction of CD82 and CD81 conjugated with eGFP
The CD82 and CD81 cDNA were amplified by PCR with appropriate oligonucleotide primers containing the restriction site for HindIII and BamHI in CD81 amplification and the restriction site for HindIII and EcoR I in CD82 amplification. eGFP expression vectors pEGFP-N1 (N1 vector) and pEGFP-C1 (C1 vector) were purchased from Clontech (Mountain View, CA, USA) and the amplified cDNAs were cloned into the plasmid’s multicloning site (MCS). CD81 or CD82 was conjugated to the N terminus of eGFP in the pEGFP-N1 plasmid (for then on: 81N1 and 82N1) and to the C-terminus of eGFP in the pEGFP-C1 plasmid (for then on: 81C1 and 82C1). The plasmids were propagated using DH5
as the host strain, employing standard procedures, and were purified employing the HiSpeed plasmid maxi kit (Qiagen Inc., Hilden, Germany).
Transient and stable transfection of plasmid DNAs
Purified plasmids 81N1, 82N1, 81C1, and 82C1 were separately introduced into RPMI 8226, CAG, PC3, and Jurkat cell lines by liposomal transfection with DMRIE-C (Invitrogen, Carlsbad, CA, USA), according to manufacturer’s instructions. In short, 100 µl OPTI-MEM I reduced serum medium containing 1.2 µl of DMRIE-C reagent and 100 µl of OPTI-MEM I medium containing 0.8 µg of DNA were added to each well. After incubation for 30 min at room temperature, 4 x 105 cells were added to each well. Following 4 h incubation at 37°C in a CO2 incubator, 0.4 ml growth medium containing 20% FBS was added to each well and incubated overnight.
For stable transfection CAG and RPMI 8226 cells were selected with 1 mg/ml and 2 mg/ml G418 (Clontech), respectively, to obtain pools of stably transfected cell lines or alternatively selected with the G418 and diluted to obtain "stable line from single cell source". Transfection efficiency was estimated according to the proportion of eGFP positive cells identified by FL1 (FITC) fluorescence. Fluorescence was analyzed by flow cytometry employing a Coulter Flow Cytometer (FACS; EPICS-XL, Beckman Coulter, Fullerton, CA, USA).
Determination of tetraspanins expression in transiently transfected MM cell lines
Transiently transfected cells were harvested, sedimented by centrifugation at 300 g, resuspended in 50 µl PBS, and assessed for CD81 and CD82 surface expression by adding the specific antibodies both phycoerythrin (PE) conjugated [CD81 from SouthernBiotech (Birmingham, AL, USA) and CD82 from Diaclone (Besancon, France)] according to the manufacturers’ instructions. IgG1-matched isotypes were used to exclude non-specific binding. Fluorescence was analyzed by flow cytometry. Experiments were repeated 3 times, and at least 10,000 events were counted in each FACS analysis. All reaction conditions and flow parameters were standardized.
Microscopic observation of transfected cells
Transfected myeloma cells excited with 488 nm wavelength by fluorescence microscope (Zeiss, Oberkochen, Germany) were watched for eGFP expression. Morphological parameters of stably transfected cells were evaluated by watching cultured cells in inverted microscope (Olympus bx60). Cells were photographed by Olympus DP70 camera.
Transfected cell survival
Transfected cells were harvested 24 h post-transfection and stained with DNA propidium iodide (PI) labeling (50 µg/ml) for 30 min. eGFP+/PI– cells analyzed by FACS were considered as the surviving cell fraction.
Analysis of apoptosis
Cells (106/1 ml HEPES buffer) were incubated with 5 µl Annexin V (PE conjugated) (MBL, Nagoy, Japan) and with 20 µl 7AAD (Beckman Coulter, Fullerton, CA, USA) for 15 min for necrotic cell staining. Positive cells were enumerated among the eGFP-positive population. Analysis was done using flow cytometry.
Caspase-3 expression by immunocytochemistry
Cells (stably transfected) were cytospinned to glass slides by centrifugation (5 min in 200 g) The slides were then washed by PBS, covered with normal blocker serum, and incubated with primary antibody (Ab) mouse antiactivated caspase 3 (Chemicon, Temecula, CA, USA) overnight. Next, the slides were incubated with biotinylated second Ab, washed, covered with horseradish peroxidase conjugated streptavidin (HRP-SA), and developed with 3-amino-9-ethyl carbazole (AEC)–chromogen (all from Zymed Laboratories, San Francisco, CA, USA). Sections were counterstained with Mayer’s hematoxylin. Isotype-matched control antibodies were used to exclude non-specific staining.
Pan caspase inhibition by ZVAD fmk
RPMI 8226 and CAG cells were incubated with 0.05 mM pan-caspase inhibitor, ZVAD fmk (R&D, Minneapolis, MN, USA) 4 h post-81N1 and –82N1 transfection. Cell survival (eGFP+/PI– cells) was determined 24 h post-transfection by flow cytometry. Untreated 81N1 and 82N1 transfected cells were considered as control.
Cell cycle
The cells were washed with PBS, resuspended, and fixated with 0.5% formaldehyde in PBS (for 15 min) followed by cell perforation (70% EtOH in PBS). The fixed cells were incubated with 300 µl staining buffer (100 µg/ml RNase A and 50 µg/ml propidium iodide) for 30 min. The DNA content in the nuclei of the cells was analyzed using flow cytometry.
Proliferation assay
Transiently transfected cells were cytospinned by centrifugation at 300 g onto SuperFrost Plus slides (Menzel-Glaser, Braunschweig, Germany). The slides were fixated with 4% paraformealdehyde and 100% methanol, blocked with 5.5% goat serum, and incubated with mixed mouse anti Ki67 (Zymed Laboratories) and chicken anti eGFP (Chemicon) primary antibodies overnight at 4°C. Next, the slides were incubated with biotinylated second Ab, washed, and covered with mixes of HRP-SA and donkey anti chicken alkaline phosphatase and developed with AEC–chromogen for Ki67 detection and with BCIP/NBT-chromogen (Chemicon) for eGFP detection. Isotype-matched control antibodies were used to exclude non-specific staining. No non-specific staining was evident (data not shown). Morphological parameters and staining of transfected/untransfected cells were evaluated by observing cells with a Nikon Labophot light microscope and photographed by Olympus DP70 camera.
Intracellular glutathione (L-
-glutamyl-L-cysteinylglycine, GSH) detection
Transfected cells were washed with PBS and then spun down. The pellet was incubated for 10 min with 0.5% formaldehyde in PBS and washed again with PBS. Thereafter, the pellet was incubated for 3 min at room temperature with 40 µM (final concentration) of mercury orange (Sigma, St. Louis, MO, USA). Cells were washed again, resuspended in PBS, and analyzed by flow cytometry (11)
.
Adhesion assay
In vitro adhesion assays were performed to evaluate the effects of 81C1 and 82C1 on the adhesive properties of RPMI 8226 and CAG cell lines with the extracellular matrix (ECM) proteins fibronectin (FN) (40 µg/ml, Biological Industries, Beit Haemek, Israel), matrigel and IGF-1 (100 ng/ml; R&D). The 24-well plates for the adhesion assays were precoated with FN or matrigel for 1 h in 37°C and then dried. 81C1, 82C1, or mock transfected cells were harvested 24 h post-transfection, centrifuged, washed with serum-free medium, and resuspended with serum-free medium to a final concentration of 1 x 106 cells/ml. Cells (0.3 x 106) were added to each well and allowed to adhere for 1 h at 37°C in humidified 5% CO2 atmosphere. The nonadherent and adherent cells were separated and quantified by FACS (percent of eGFP+ and eGFP– cells in the adherent and nonadherent cells fractions). We calculated the ratio between percent of adherent eGFP+ cells and percent of adherent eGFP– cells in each treated well. Ratio = 1 indicated no differences in adherence of eGFP+ and eGFP– cells in the well; ratio >1 indicated an increase in adherence of eGFP+ cells; ratio <1 indicated a decreased adherence of eGFP+ cells. All treatments were compared with the corresponding mock transfected cells.
Migration assay
RPMI 8226 and CAG transfected cells were centrifuged 24 h post 82C1 and 81C1 transfection, resuspended in 200 µl OPTI-MEM I medium, and seeded in the upper compartment of transwell (Corning Inc., Acton, MA, USA; 2.5x105 cells per well). The lower chamber was precoated with FN, as described previously, and covered with 700 µl medium contained 10% FBS. After 24 h, cells that migrated through the membrane to the lower chamber were separated from the nonmigrating cells. The nonmigrating cells in the top chamber and the migrating cells present in the bottom chamber were separated and quantified by FACS (percent of eGFP+ and eGFP– cells in the migrating and nonmigrating cells fractions). We calculated the ratio between percent of migrating eGFP+ cells and percent of migrating eGFP– cells in each treated well. Ratio = 1 indicated no differences in migration of eGFP+ and eGFP – cells in the well; ratio >1 indicated an increase in migration of eGFP+ cells; ratio <1 indicated a decreased migration of eGFP+ cells. All treatments were compared to the corresponding mock transfected cells.
Gelatin zymography
Media of stable cells cultures were collected for gelatinase activity. Aliquots (20 µl) of the media were electrophoresed at nonreducing conditions in 10% polyacrylamide gels containing 1 mg/ml gelatin type A (Sigma). Gels were washed twice in 2.5% Triton X-100 for gelatinase renaturation and incubated overnight in 50 mM Tris-HCl (pH 7.5) and 5 mM CaCl2. Coomassie blue staining followed by destaining allowed visualization of clear lysis zones against a blue background. Band intensity was calculated for each example, compared with positive control [standard zymography activated and proactivated MMP-9 (Chemicon)] in each gel assay by employing Gel Doc 2000 (Bio-Rad Laboratories, Hercules, CA, USA).
Statistical analysis
Student’s paired t tests were employed in analysis of differences between cohorts. An effect was considered significant when P-value was equal to or less than 0.05. All experiments were conducted three to seven separated experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). The initial biological implication of the fusion protein was demonstrated by the differential cellular distribution between mock and tetraspanins transfected cells (Fig. 1A
). In addition, CD81 and CD82 up-regulation was determined by FACS (Fig. 1B
). Stably transfected cells were selected, yet while the 81N1/82N1 transfected cells (single cell selection and pools) developed into small colonies (50–100 cells) and died massively at that stage (Fig. 2
), stably transfected descendents with mock vectors were established in both cell lines. Long-term culturing of the 81C1/82C1 stably transfected cell lines displayed a time-dependent decrease in eGFP expressing cells (data not shown). Thus, we hypothesized that the induced expression of the tetraspanin had a deleterious effect on the myeloma cell lines and was negatively selected. Therefore, most of the presented results originate from experiments conducted on transiently transfected cells.
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CD81 and CD82 cloned in pEGFP-N1 vector induce MM cell death
Transient transfection of 81N1 and 82N1 into MM cell lines had a deleterious effect evidenced in reduced fractions of surviving cells (eGFP+/PI–) (Fig. 3
A) and significant increase of necrotic cell death (Annexin V+/7AAD+) compared to mock (Fig. 3B
) (P<0.05). Transfection of 81C1 and 82C1 did not affect cell survival (Fig. 3A
).
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The extent of induced cell death in the CAG population was higher than in RPMI 8226 cells (P<0.05). Moreover, 81N1 transfection resulted in higher death rates than 82N1 in both cell lines, yet the difference was statistically significant in CAG alone (P<0.05) (Fig. 3B
). In the 82N1 stably transfected CAG at the stage of ensuing cell death, elevated levels of caspase 3 were observed (P<0.05) (Fig. 4
A). However, treatment with the pan caspase inhibitor, ZVAD fmk failed to prevent the tetraspanin-induced cell death, which indicated that the death was caspase-independent (Fig. 4B
).
|
Recent publications reported of CD81 and CD82 overexpression in various cell systems, including PC3 (12
, 13)
and Jurkat (14)
. Hence, we decided to corroborate our findings in these cell line models. In accordance to previous publications CD81 and CD82 are expressed in Jurkat cells (15)
, while PC3 cells express CD81 (data not shown) but not CD82 (12)
.
Indeed, 81N1 and 82N1 reduced cell survival in both lines (P<0.05) (Fig. 5A
), indicating that this effect is not limited to myeloma. While the death of PC3 cells was necrotic in nature (Fig. 5B
), the Jurkat cell line demonstrated a combination of apoptotic and necrotic death modes (Fig. 5B
). Interestingly, Jurkat cells were also susceptible to 81C1- and 82C1-instigated cell death as well (P<0.05) (Fig. 5A, B
).
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81N1 and 82N1 transfection reduced cell proliferation but did not affect cell cycle
In 81N1 and 82N1 transfected CAG and RPMI 8226 reduced staining with Ki67 proliferation marker was demonstrated 24 h post-transfection (Fig. 6
). Yet, the reduced cell proliferation was not accompanied by changes in cell cycle (data not shown). No changes were found in proliferation or cell cycle of the MM cell lines transfected with 81C1/82C1 constructs.
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Elevated glutathione level in 82N1 and 81N1 transfected cells
In our study, augmented glutathione levels were observed in the 81N1 and 82N1 transfected MM cell lines (Fig. 7
). Moreover, the elevation of glutathione was in concordance with the death rate: 81N1 transfected CAG and RPMI 8226 displayed + 38.1%, +50.5% increases, respectively, whereas 82N1 transfected CAG and RPMI 8226 exhibited + 20.2%, +17.2% increase rates, respectively (compared to mock).
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81C1 and 82C1 reduce MM cell adhesion, migration, and invasion
Adhesion
At 24 h post-transient transfection of CAG and RPMI 8226 cells with 81C1 or 82C1 vectors, cells were harvested and seeded in tissue culture plates with (I) uncoated wells (II) wells precoated with FN (III) uncoated wells with IGF-I supplementation (IV) wells precoated with FN and supplementation of IGF-I and (V) wells precoated with matrigel. Both eGFP+ and eGFP– adherent cells enumerated by FACS. Results presentation is detailed in Materials and Methods. 82C1 attenuated RPMI 8226 cell adherence in uncoated (I) (P<0.05), and FN precoated (II) (P<0.05) wells but did not modify cell adherence to matrigel coated wells (V) (Fig. 8
A). However, 82C1 transfected CAG cells displayed differential adherence with FN and matrigel precoated plates only (P<0.05) (Fig. 8B
). IGF-I had a deleterious effect on 82C1 transfected RPMI 8226 cell adhesion with and without FN (Fig. 8A
). Yet, when applied in combination with FN to 82C1 transfected CAG it abrogated the negative effect of FN alone and was not different than mock transfected cells (Fig. 8B
).
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The effect of 81C1 expression on adhesion of MM cells was less significant compared with 82C1. RPMI 8226 adhered less to FN with/without IGF-I when transfected with 81C1 (P<0.05) (Fig. 8A
). CAG cell adhesion was diminished by the 81C1 expression only on matrigel-coated wells (P<0.05) (Fig. 8B
).
Migration
Transfected cells were seeded into transwell chambers, and 24 h later both eGFP+ and eGFP– cells in the bottom section were enumerated by FACS. Results presentation is detailed in Materials and Methods. 82C1 expression significantly reduced the number of migrating cells in both cell lines (P<0.05), whereas 81C1 had deleterious effects on cell migration in RPMI 8226 cells only (P<0.05) (Fig. 9
).
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MMP9 secretion
We determined the invasion cell potential of MM cell lines by measuring MMP-9 activity levels secreted from RPMI 8226 and CAG cells into culture media. Down-regulation of MMP-9 activity was measured in both cell lines expressing 82C1 (–18% and –38% in RPMI 8226 and in CAG, respectively) (P<0.05) but not in those expressing 81C1 (Fig. 10
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In our study we did not detect any cell cycle arrest, thus we deduce that the tetraspanin overexpression reduced the cycling rate and culminated in cell death. Addition of pan caspase inhibitor ZVAD fmk to the transient transfected cells did not prevent the cell death, substantiating that the mode of cell death is caspase-independent. The up-regulation of activated caspase-3 we determined in the CAG-82N1 stable cell clones is interesting in the situation of the caspase-independent death of the cells. Indeed, caspase 3 activation can be assessed in the context of recent publications. A study described necrotic death accompanied by caspase-3 cleavage that could not be diminished by pan-caspase inhibitors (17)
. Moreover, the existence of caspase-3 activation was also shown to be necessary for proteolytic activity other than apoptosis (18)
.
In our study, MM cell death was characterized by elevation of glutathione levels following 81N1 and 82N1 transfection. Glutathione is an H2O2 scavenger and a part of the antioxidant cellular redox formation (19
, 20)
. Usually reduced-glutathione (GSHv) levels result in elevated H2O2 and cause cell death (19)
. We, however, observed an up-regulation of glutathione, which may indicate that the cells are trying to cope with elevated H2O2 levels, but the final outcome of cell death indicates that the antioxidant activity is not sufficient. We surmise that it is safe to say that a fatal deregulation of the redox balance in the tetraspanin transfected cells occurs. Indeed, a recent publication (12)
showed a down-regulation of glutathione levels in CD82 transfected HeLa cells, which culminated in cell death. Taken together, it can be concluded that tetraspanins are involved in glutathione cellular equilibrium but that the mechanism of their involvement has yet to be deciphered. Moreover, glutathione is also implicated in cell signal transduction, thus additional research must determine whether the tetraspanin-induced glutathione elevation is associated with activated signaling pathways or related only to oxidant neutralization.
Cell death by tetraspanins was previously reported in other publications: apoptosis by CD82 in PC3, HeLa, MCF-7 (12)
, IdlD (Chinese hamster ovary cells) (13)
, pancreatic cells (21)
; and by CD81 in HeLa (12)
.
Complying with the publication of the Schoenfeld group (12)
we demonstrated that 81N1 and 82N1 induced PC3 cell death as well; however, we observed a necrotic form of death, whereas Schoenfeld et al. described an apoptotic mode of death by CD82 with an untagged vector. We also transfected 81N1/81C1 and 82N1/82C1 into Jurkat cells and observed a combination of necrotic and apoptotic death by both tetraspanins in the two cloning orientations. A recent publication showed that 82N1 transfection into an SV40 transformed Jurkat cell line did not influence cell viability (14)
. We postulate that the SV40 transformation, which causes a constitutive Akt phosphorylation (a known survival factor) (22
23
24)
, activates a survival signal that is resistant to the effect of tetraspanins. Studies addressing the role of Akt in the tetraspanin signaling pathway in myeloma are currently underway in our laboratory.
Tetraspanins constructed into the C1 vector did not induce cell death, yet they generally had a deleterious effect on cell adhesion, motility, and invasion. 82C1 introduction into CAG and RPMI 8226 cultured on FN caused a decrease in the number of adherent and migrating cells. These phenomena were also observed in 81C1 transfected RPMI 8226 cells. Moreover, the down-regulation of migration was in concordance with reduced adhesion (on FN). IGF-I, as expected from its role as a cell adhesion initiator (25)
, diminished (RPMI 8226) or abrogated (CAG) the deleterious effect of 81C1 and 82C1 on cell adhesion. The involvement of tetraspanins in cell adhesion is attributed to their interactions with ß1 integrins (26)
. Indeed, it was reported that the interaction of CD82 with 6 integrin prevented its association with other signaling proteins and thus inhibited cell adhesion (27)
.
MMPs degrade ECM and facilitate tumor cell invasion. Specifically, MMP-9 is frequently up-regulated in many malignancies and is correlated with myeloma progression. Also, current literature portrays MMP-9 as the primary metalloproteinase in myeloma (28)
. We showed that 82C1 overexpression caused reduction of secreted MMP-9. Indeed, tetraspanins have been reported to up- and/or down-regulate MMPs depending on MMP-type, specific tetraspanin, and/or tissue (29
, 30)
. Yet, no previous study has attributed CD82 with an effect on MMP. The combined results described in here support the multifaceted involvement of CD81 and CD82 in the invasive potential of the myeloma cells.
This study delineates, for the first time, the importance of CD81 and CD82 expression to MM cell survival and their negative effects on cell adhesion, motility, and invasion. The differential effects of the pEGFP-N1 and pEGFP-C1 tetraspanin vectors can be attributed to specific terminus functions (or lack of) or critical variation of fusion protein conformation and warrants additional study.
The down-regulation of tetraspanins in myeloma and wide spectrum of effects induced by their re-expression underscore their essential role in cellular functions. Moreover, the deleterious effect of tetraspanins on adhesion/migration/invasion/viability supports their role as tumor suppressors.
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ACKNOWLEDGMENTS |
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Received for publication July 31, 2006. Accepted for publication October 25, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6-mediated cell adhesion. J. Biol. Chem. 280,3346-3354
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
