|
Article |
Address correspondence to F.M. Watt, Keratinocyte Laboratory, CR-UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, England. Tel.: 44 20 7269 3528. Fax: 44 20 7269 3078. email: fiona.watt{at}cancer.org.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: keratinocyte; apoptosis; differentiation; Akt; extracellular matrix
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Integrin expression is frequently perturbed in squamous cell carcinomas (SCCs), which are tumors of stratified squamous epithelia (Mercurio and Rabinovitz, 2001; Watt, 2002). One such change is an up-regulation of vß6. In normal epidermis,
v forms a heterodimer exclusively with ß5 (Pasqualini et al., 1993). In hyperproliferative stratified squamous epithelia, both
vß5 and
vß6 are expressed (Breuss et al., 1995; Haapasalmi et al., 1996). In SCCs,
vß6 is up-regulated, and this is often correlated with a down-regulation of
vß5 expression (Jones et al., 1997; Regezi et al., 2002). The down-regulation of
vß5 in association with expression of
vß6 probably reflects a hierarchy in the preference of
v to heterodimerize with different ß subunits (Koistinen and Heino, 2002).
There are several ways in which up-regulation of the vß6 integrin can affect keratinocyte behavior.
vß6 binds the latency-associated peptide derived from latent TGFß and activates TGFß (Munger et al., 1999). Expression of
vß6 increases epithelial cell motility and invasion (Thomas et al., 2001b; Ramos et al., 2002), stimulates cell proliferation (Ahmed et al., 2002), and inhibits fibronectin matrix assembly (Ramos et al., 2002). The effects on growth and invasion may reflect the abilities of
vß6 to activate Erk2 MAPK (Ahmed et al., 2002) and to up-regulate or activate matrix metalloproteinases (Agrez et al., 1999; Thomas et al., 2001a; Ramos et al., 2002).
We have previously described a cell line that provides an excellent model system for studying the significance of different v integrins in SCCs. The H357 cell line is derived from a human SCC of the tongue and lacks expression of
v integrins (Sugiyama et al., 1993). When the
v subunit is transfected into H357 cells and
v-positive clones selected by multiple rounds of FACS®,
v is expressed on the cell surface as
vß5. Expression of
vß5 is correlated with inhibition of anchorage-independent growth and induction of terminal differentiation in H357 cells (Jones et al., 1996). In the present experiments, we have uncovered a previously unrecognized function of
vß5 in promoting suspension-induced apoptosis, known as anoikis (Frisch and Francis, 1994), and show that when
vß5 is replaced at the cell surface with
vß6, anoikis is prevented. These observations suggest that up-regulation of
vß6 may confer a survival advantage on cells in SCCs.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell surface expression of v and
4 was examined by flow cytometry (Fig. 1). As reported previously (Sugiyama et al., 1993), parental H357 cells did not express detectable surface levels of
v (Fig. 1 a). Transduction with the
v retroviral vector resulted in higher cell surface levels of the integrin subunit (Fig. 1 b) than achieved previously by transfection and clonal selection (Fig. 1 c). As expected, parental cells did not express the
4ß1 integrin (Fig. 1 d). The level of cell surface expression of the
4 subunit after retroviral transduction (Fig. 1, e and f) was the same whether or not the cells were also transduced with the chick ß1 subunit (Fig. 1 f), indicating that the endogenous pool of ß1 subunits was not limiting. Introduction of the
v or
4 integrin subunits had no effect on cell surface levels of any of the endogenous ß1 integrins (Fig. 1, gi, and not depicted) nor on levels of
6ß4 (Fig. 1, jl).
|
To examine whether or not the vß5 heterodimer expressed by retrovirally transduced H357 cells was functional, adhesion assays on vitronectin were performed. Up to 90% of H357 cells expressing
vß5 adhered to vitronectin, compared with <5% of the parental cells or H357 cells transduced with the
4 integrin subunit (Fig. 2 a). H357 cells transduced with the
v integrin subunit spread rapidly on vitronectin, and within 20 min
vß5 was detected in focal adhesions (Fig. 2, bd).
vß5 did not localize to focal adhesions in cells plated on fibronectin (Fig. 2 e). Conversely, ß1 integrins localized to focal adhesions when cells were plated on fibronectin (Fig. 2 f) but not on vitronectin (Fig. 2 g), suggesting that
vß1 heterodimers did not form.
|
|
vß5 induces anoikis of H357 cells
Next, we investigated whether or not expression of vß5 induced anoikis (Frisch and Francis, 1994). We determined the proportion of cells with a sub-G1 DNA content, as a measure of the number of apoptotic cells (Frisch, 1999b). We compared parental H357 cells, cells transduced with the empty retroviral vector, cells transduced with
v, and cells transduced with
4.
Typical flow cytometry profiles are shown in Fig. 3 a, and data pooled from six experiments are shown in Fig. 3 b. The proportion of cells with a sub-G1 DNA content was <3% in freshly harvested cells. In parental cells and cells transduced with 4 or empty vector, the proportion increased to almost 10% by 48 h and 20% by 72 h in suspension. Cells transduced with
v had a similar level of apoptosis to controls after 24 h in suspension, but by 48 h the proportion of apoptotic cells had increased to almost 30%, and to over 50% by 72 h. The increased apoptosis of H357 cells transduced with
v compared with controls was statistically significant (P = 0.008 at 72 h; t test).
|
Anoikis is inhibited by integrin ligation of suspended cells or by treatment with caspase inhibitors (Frisch, 1999b). Therefore, we examined the effects of these treatments on H357 cells expressing vß5 (Fig. 3 c). The cells were held in suspension for 48 or 72 h in medium alone (control) or containing DMSO (caspase inhibitor solvent), the pan caspase inhibitor z-VADfmk (100 µM), vitronectin (100 µg/ml;
v ligand), or the
v function-blocking antibody 13C2 (100 µg/ml; Davies et al., 1989). At both time points, z-VAD-fmk and vitronectin substantially reduced the proportion of apoptotic cells. 13C2 also had an inhibitory effect, albeit slightly smaller.
We conclude that unligated vß5 expression in H357 cells leads to an increase in suspension-induced apoptosis and that this is the likely mechanism by which anchorage-independent growth is inhibited.
Effect of overexpressing v in cells with normal endogenous levels of
vß5
To investigate whether or not vß5-induced anoikis was specific to H357 cells or a more general phenomenon, we transduced a second SCC line, SCC4, and primary human keratinocytes with the
v or
4 retroviral vectors (Fig. 3, dg). SCC4 has a similar level of endogenous
v integrins to primary keratinocytes (Fig. 3, d and f; Levy et al., 2000), but is heterozygous for an activating ß1 integrin mutation (Evans et al., 2003). The levels of
v expression achieved by retroviral transduction were higher than the endogenous levels (Fig. 3, d and f) and equivalent to that of
v-transduced H357 cells (Fig. 1 b).
Fig. 3 h shows pooled data from three experiments in which the proportion of apoptotic SCC4 cells was compared immediately after detachment from the culture dish (0 h) or after suspension for 24 or 48 h. At both 24 and 48 h, the proportion of apoptotic cells was significantly higher in cells transduced with v than in parental SCC4 or SCC4 transduced with the
4 retroviral vector (P < 0.05).
It has previously been reported that when primary human keratinocytes are placed in suspension they undergo terminal differentiation as opposed to anoikis (Gandarillas et al., 1999). Consistent with those observations, the proportion of primary keratinocytes in suspension with a sub-G1 DNA content did not exceed 15%, even after 48 h (Fig. 3 i). The proportion of apoptotic cells was not significantly different when parental keratinocytes were compared with keratinocytes transduced with 4 or
v retroviral vectors (Fig. 3 i). The proportion of cells that underwent terminal differentiation was also unaffected by overexpression of
v or
4 (unpublished data).
We conclude that in the SCC lines examined elevated expression of v stimulated anoikis independent of whether or not the parental cells expressed
v. However, overexpression of
v in primary keratinocytes was not sufficient to induce anoikis.
Role of PI3 kinase activation in anoikis
Signaling pathways known to protect cells from anoikis include FAK activation leading to Erk MAPK phosphorylation and PI3 kinase activation resulting in activation of PKB/Akt (Frisch et al., 1996). There is conflicting evidence regarding c-Jun kinase and p38 MAPK (Frisch et al., 1996; Khwaja and Downward, 1997). To examine the role of these pathways in anoikis of H357 cells, we added the PI3 kinase inhibitor LY294002, the MEK1/2 inhibitor UO126, and the p38MAPK inhibitor SB203580 to H357 cells transduced with v or
4 and measured the proportion of apoptotic cells after 72 h in suspension (Fig. 4 a). z-VAD-fmk was added as a positive control to block anoikis (Fig. 4 a). None of the inhibitors tested had any effect on the proportion of
vß5-expressing cells that underwent apoptosis. However, the PI3 kinase inhibitor increased apoptosis of
4-expressing cells to the level of cells expressing
vß5 (Fig. 4 a).
|
The proapoptotic effect of vß5 is not shared with
vß6 and is mediated by the ß5 cytoplasmic domain
Because up-regulation of vß6 is a feature of many SCCs, we investigated whether or not this integrin, like
vß5, promoted anoikis. H357 cells transduced with the
v retroviral vector were infected with a second vector that encoded the ß6 integrin subunit. Introduction of ß6 had no effect on cell surface levels of
v (Fig. 5 a; compare Fig. 1 b). However, cells no longer expressed surface
vß5 (detected with P1F6 antibody); instead all of the
v on the cell surface was now in a heterodimer with the ß6 subunit (detected with 10D5 antibody; Fig. 5 a).
|
In contrast to cells expressing vß5, cells expressing
vß6 did not undergo anoikis and had the same sub-G1 DNA profiles as parental H357 cells (Fig. 5 f) and cells transduced with the
4 integrin subunit (Fig. 5 g). Whereas cells expressing
vß5 did not activate Akt in suspension, cells expressing
vß6 did activate Akt (Fig. 5 h) and the kinetics were similar to parental cells and cells expressing
4 (Fig. 4 b and not depicted). Akt activation in
vß6-expressing cells was blocked by treatment with the PI3 kinase inhibitor LY294002 (Fig. 5 h).
To investigate whether or not the cytoplasmic domain of the ß5 subunit conferred sensitivity to anoikis, we constructed a chimeric integrin subunit, consisting of the ß6 extracellular and transmembrane domains and the ß5 cytoplasmic domain (ßX6C5). H357 cells were doubly infected with v and ßX6C5 retroviral vectors, and cells were examined by flow cytometry with an
vß6-specific antibody (Fig. 5 b). Cells transduced with ßX6C5 alone did not express the chimera on the cell surface, confirming that it only heterodimerized with
v (unpublished data).
Cells were placed in suspension for 72 h, and the proportion of apoptotic cells was compared in parental H357 cells and cells expressing vß5,
vß6, or
vßX6C5. The results of one experiment are shown in Fig. 5 f, and data pooled from three separate experiments are shown in Fig. 5 g. H357 cells expressing
vßX6C5 were as susceptible to anoikis as cells expressing
vß5. We conclude that the cytoplasmic domain of ß5 mediates the proapoptotic effect of
vß5.
The ability of H357 cells to avoid anoikis again correlated with their ability to activate Akt in suspension (Fig. 5 i). Parental cells and cells expressing vß6 activated Akt, whereas cells transduced with
v (expressed on the cell surface as
vß5) or
vßX6C5 did not.
Activation of Akt can overcome the proapoptotic effect of vß5
If vß5 expression triggers anoikis by preventing activation of Akt, then constitutive activation of Akt should allow
vß5-expressing cells to survive in suspension. Two Akt constructs were introduced into H357 cells. The first construct, M+Akt:ER*, has a myristoylation targeting sequence fused to the NH2 terminus of a constitutively active form of Akt lacking the PH domain (myrAkt del4-129). At the COOH terminus is a modified form of the hormone-binding domain of the mouse estrogen receptor (ER) that binds 4-hydroxytamoxifen (OHT) but is refractory to estrogen. In response to OHT, M+Akt:ER* is rapidly recruited to the plasma membrane where it is activated by local PI3 kinases. The second construct, A2M+Akt:ER*, acts as an inactive control because the myristoylated glycine at the second amino acid position has been converted to alanine to eliminate the membrane-targeting function (Kohn et al., 1998). M+Akt:ER* and A2M+ Akt:ER* were expressed in retroviral vectors and introduced into parental and
v-expressing H357 cells. Adherent cultures were treated with OHT for 1 h, lysed, and examined by Western blotting. An antibody to the mutant ER detected expression of the Akt constructs at equal levels in all cell populations (Fig. 6 a). The level of endogenous serine 473phosphorylated Akt was also the same in all cells (Fig. 6 a). However, only cells expressing M+Akt:ER* had a second band detected by anti-phosphoAkt, which migrated at 97 kD, the size of the AktER fusion protein (Fig. 6 a). H357 parental cells and cells expressing
v or
4 were transduced with the active or inactive Akt constructs and placed in suspension for 72 h in the presence of OHT (Fig. 6, b and c). Parental H357 cells and
4-expressing cells had a low level of anoikis that was not influenced by Akt activation. H357 cells expressing
vß5 and the inactive Akt construct underwent anoikis, and the proportion of apoptotic cells was equivalent to that seen in cells expressing
vß5 alone (Fig. 6 c). H357 cells expressing active Akt were protected from anoikis and had the same low level of apoptotic cells as H357 parental cells and cells expressing
4 (Fig. 6, b and c). The conclusions from the flow cytometry experiments were confirmed by evaluating nuclear morphology of cells stained with Hoechst 33258 (unpublished data). The level of serine 473phosphorylated Akt was compared in
v-expressing H357 cells transduced with M+Akt:ER* or A2Akt:ER* after 72 h in suspension (Fig. 6 d). Cells expressing the inactive construct had no activation of AktER and either low or no phosphorylation of endogenous Akt. In contrast, both the myristoylated construct and endogenous Akt were activated in cells expressing M+Akt:ER*.
|
Suspension experiments were performed in the presence of the general caspase inhibitor z-VAD-fmk (Fig. 4 a and Fig. 7 a), the caspase 8 specific inhibitor z-IETD-fmk, and the caspase 9 inhibitor z-LEHD-fmk (Fig. 7 a). All three drugs inhibited apoptosis of vß5-expressing cells to the same extent (Fig. 7, a and b). This finding suggested that the intrinsic pathway was involved in anoikis.
|
Inhibition of caspase 8 increases Akt activity
We investigated potential mechanisms by which Akt phosphorylation was down-regulated when vß5-expressing H357 cells were held in suspension for 72 h. PTEN is a negative regulator of Akt-dependent cell survival (Stambolic et al., 1998); however, there was no difference in PTEN levels in parental cells or cells expressing
vß5 or
vß6 (Fig. 8 a). In addition, there was no difference in expression of PI3 kinase, as judged by the level of the p110
subunit detected on Western blots (Fig. 8 b), nor in the total level of Akt (Fig. 8 c).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ability of integrin ligation to suppress apoptosis is well established, and several different mechanisms have been reported, reflecting a degree of cell type and integrin specificity (for review see Jost et al., 2001; Miranti and Brugge, 2002). Ligation of integrins can induce antiapoptotic proteins, such as Bcl-2 (Matter and Ruoslahti, 2001), or suppress proapoptotic proteins, such as Bax (Gilmore et al., 2000; Valentijn et al., 2003). Ligation of 5ß1 or
vß3 increases Bcl-2 expression through NF
B activation (Scatena et al., 1998). Integrin activation of FAK leads to increased expression of inhibitors of apoptosis proteins, which bind to and inhibit executioner caspases (Sonoda et al., 2000).
Previous work on anoikis has demonstrated the involvement of death receptor signaling and the subsequent activation of the extrinsic death pathway via caspase 8 activation (Frisch, 1999a; Rytömaa et al., 2000). vß5-mediated anoikis could not be inhibited with dnFADD, suggesting that unlike anoikis of endothelial cells and simple epithelial cells (Frisch, 1999a; Rytömaa et al., 1999; Hood et al., 2003), SCC anoikis is independent of death receptor signaling. In endothelial cells,
vß5 ligation protects against FADD-mediated apoptosis (Hood et al., 2003), demonstrating that the same integrin can regulate apoptosis by distinct mechanisms, dependent on cellular context.
vß5-mediated anoikis could be blocked with inhibitors of caspase 8 and 9. Caspase 8 can be activated independently of the extrinsic death pathway in cells with unligated integrins (Stupack et al., 2001). Caspase 8 cleaves Bid (a Bcl-2 family member), triggering mitochondrial cytochrome c release and activation of caspase 9 (Rytömaa et al., 2000). Caspase 9 may then lead to further cleavage and activation of caspase 8, hence amplifying the signal (Rytömaa et al., 2000).
It has previously been reported that whereas unligated ß1 and ß3 integrin subunits can directly activate caspase 8, ß5 does not (Stupack et al., 2001). In agreement with this report, we were unable to coimmunoprecipitate vß5 or
vß6 with caspase 8 in H357 cells under a variety of conditions (unpublished data). In H357 cells expressing
vß5 it is more likely that the ß5 cytoplasmic tail activates caspase 8 indirectly via effects on the cytoskeleton. In some dying cells, actin, integrins, and caspase 8 have been seen to colocalize in complexes that lack death receptors and FADD (Stupack et al., 2001), and local accumulation of caspase 8 is sufficient to trigger apoptosis. The mechanism of the association of caspase 8 with the cytoskeleton is not known, but newly described proteins Hip and Hippi, homologous to cytoskeleton linker proteins talin and myosin, have been found that include "pseudo-death effector domains" (Gervais et al., 2002).
In contrast to v-negative H357 cells and cells expressing
vß6, cells that expressed
vß5 did not activate Akt in suspension. The pro-survival function of Akt is well established. Activated Akt can phosphorylate and inhibit several different proapoptotic proteins including BAD (Datta et al., 1997) and caspase 9 (Cardone et al., 1998). Active Akt also induces NF
B (Kane et al., 1999), which reduces the release of cytochrome c from mitochondria (Kennedy et al., 1999). Akt is negatively regulated by the phosphatase PTEN, and a lack of PTEN leads to increased resistance to apoptosis (Lu et al., 1999). However, there were no differences in PTEN levels in parental H357 cells or cells expressing
vß5 or
vß6.
The mechanism by which unligated vß5 triggers anoikis requires activation of caspases 8 and 9 and suppression of Akt activity. Because there was no reduction in total Akt when
vß5-expressing H357 cells were held in suspension, caspase-dependent cleavage of Akt (Bachelder et al., 2001) does not appear to be involved. Nevertheless, inhibition of caspase 8 did lead to an increase in phosphoAkt, placing caspase 8 upstream of the suppression of Akt activation. Inhibition of caspase 9 did not increase phosphoAkt, leading us to speculate that Akt is upstream of caspase 9 (Cardone et al., 1998).
One striking difference between primary human keratinocytes and SCC cells is that primary keratinocytes undergo terminal differentiation and not anoikis when held in suspension (Gandarillas et al., 1999). Moreover, when unligated integrins are expressed in the suprabasal layers of the epidermis, apoptosis is not stimulated (Carroll et al., 1995). When v was overexpressed in primary human keratinocytes they retained the ability to terminally differentiate and did not undergo anoikis. It seems possible that the selection pressure placed on clones of
v-transfected H357 cells described previously selected for rare cells that had the ability to terminally differentiate rather than apoptose (Jones et al., 1996).
The similarities between apoptosis and keratinocyte terminal differentiation have previously been discussed and include inhibition of suspension-induced differentiation by integrin ligation (Levy et al., 2000) and by caspase inhibitors (Allombert-Blaise et al., 2003). Akt activation plays a role in driving the later stages of keratinocyte terminal differentiation (Janes et al., 2004). We speculate that by preventing Akt activation, vß5 acts as a fail-safe device, triggering anoikis in SCC cells that have lost the ability to differentiate. Resistance to anoikis can potentially enhance epithelial neoplasia by allowing cells to proliferate in the absence of attachment to extracellular matrix and to leave their normal environment and travel to distant body sites, thereby forming metastases. Up-regulation of
vß6 is a novel mechanism by which tumor cells can avoid anoikis.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Retroviral vectors
To generate vpBabe puro, human
v cDNA (provided by J. Loftus, Scripps Research Institute, La Jolla, CA) was blunted and inserted into SnabI cut, shrimp alkaline phosphatasetreated pBabe puro vector (Levy et al., 1998) using T4 ligase.
4pBabe puro and ß6pBabe puro were gifts from J. Marshall and I. Hart (Cancer Research UK, London, UK; Thomas et al., 2001b). M+Akt:ER* and A2M+Akt:ER (Kohn et al., 1998; Mirza et al., 2000) in retroviral vector pWZLneo were gifts of L.M. Martins and J. Downward (Cancer Research UK, London, UK). eGFP-dNFADD-AU1 was excised from pcDNA3-AU1-NF4D (a gift of J. Downward; Rytömaa et al., 1999) with NheI and BamHI, blunted and inserted into SnabI cut, shrimp alkaline phosphatasetreated pBabe puro using T4 ligase.
The chimeric ß6/ß5 integrin subunit (ßX6C5) was constructed as follows. The ß6 extracellular and transmembrane domain was made by PCR using ß6pBabe puro as a template. The primers used had the BamHI and EcoRI sites added and were as follows: 5'-GGGATCCGCCACCATGGGGATTGAACTGCTTTGCCTGTTCTT-3'; 3'-GGAATTCTTCCAGATGCACAGTA- GGACAACCCCGATGA. The resulting ß6 cytoplasmic domain-deleted PCR product (ß6C) was cloned into pCR-4-TOPO, cut with EcoRI and BamHI, and ligated into pBabe puro. The cytoplasmic domain of ß5 was purchased as part of an I.M.A.G.E. clone (7160-k21; Geneservice) and generated by PCR using primers that had an EcoRI site at the 5' prime end and SalI at the 3' end. The primers were as follows: 5'-GGAATTCTGCTTGTCACCATCCACGACCGGAGGGAGTT-3'; 3'-GAGTCGACTCAGTCCACAGTGCCATTGTAGGATTTGTTG. The confirmed sequence was cut using EcoRI and SalI and ligated into ß6
C pBabe puro giving the ßX6C5 chimeric molecule, but with an additional EcoRI site. This site was removed using Mung Bean Nuclease. The cDNA was then religated, bringing the chimeric integrin subunit correctly into frame.
Retroviral transduction
Ecotropic Phoenix packaging cells were transiently transfected with retroviral vectors, and virus-containing supernatants were used to infect AM12 packaging cells, as described previously (Legg et al., 2003). 6085% of AM12 cells transduced with retrovirally encoded integrin subunits had high surface expression of the integrins following puro selection; these cells were selected by FACS® to achieve high expression in >95% of the population (Levy et al., 1998). Stably transduced AM12 cells were cultured with 2.5 µg/ml puromycin or, in the case of the Akt retroviral vectors, 2 mg/ml G418 (Geneticin; GIBCO BRL).
SCC cells and primary keratinocytes were transduced with retroviral vectors either by coculture with AM12 cells (Levy et al., 1998) or by incubation with AM12 supernatant (Legg et al., 2003). 7085% of epithelial cells had high surface expression of the retrovirally encoded integrin subunits after puro selection; these cells were further enriched by FACS® as described for AM12 cells.
When cells were transduced with two retroviral vectors, selection was either with puromycin and FACS® or with puromycin and neomycin. SCC and primary keratinocytes transduced with retroviral vectors were cultured with 1 µg/ml puromycin and/or 2 mg/ml G418, as appropriate.
Antibodies
The following mAbs to human integrins were used: HAS4 (2ß1), VM-2 (
3ß1), 7.2R (
4; a gift from J. Marshall and I. Hart), 13C2 (
v subunit), PIIW7 (
v subunit), P1D6 (
5ß1; Chemicon), GoH3 (
6 integrins), P1F6 (
vß5), AB1932 (
vß3; Chemicon), 23C6 (
vß3), 10D5 (
vß6; Chemicon), MPF410 (
6 integrins), P5D2 (ß1 integrins), and 3E1 (ß4 integrin subunit). We used JG22 to detect chick ß1 integrins. Actin was detected with AC40 (Sigma-Aldrich), transglutaminase 1 with B.C1 (a gift of R. Rice, University of California, Davis, Davis, CA), and involucrin with SY5. An antibody to AU1 was purchased from Covance and anti-p110
PI3K was purchased from BD Biosciences. Rabbit antibodies were also purchased as follows: ER (HC20; Santa Cruz Biotechnology, Inc.), serine 473phosphorylated Akt (Biosource International; Cell Signaling Technology), total Akt (Cell Signaling Technology), Erk1/2 (Cell Signaling Technology), phospho-Erk1/2 (Thr202/Tyr204; Cell Signaling Technology), Erk2 (SC-1647; Santa Cruz Biotechnology, Inc.), and PTEN (Cascade Biologics, Inc.). Rabbit antibodies against ß5 and ß6 used for Western blotting were purchased from Calbiochem. Species-specific secondary antibodies conjugated to Alexa 488 or Alexa 594 were purchased from Molecular Probes, and HRP-conjugated second antibodies were purchased from Amersham Biosciences.
FACS®, flow cytometry, and immunofluorescence staining
Live cells were labeled with antibodies diluted in PBSABC and sorted into FAD, 10% FCS, and HICE medium using a FACSVantageTM machine (Becton Dickinson), as described previously (Levy et al., 1998). Involucrin and transglutaminase 1 expression was examined by flow cytometry of PFA-fixed, saponin-permeabilized cells, as described previously (Gandarillas et al., 1999), using a FACScanTM machine (Becton Dickinson).
DNA content of ethanol-fixed cells stained with 5µg/ml propidium iodide in the presence of RNase A (50 µg/ml) was determined with a FACSCalibur flow cytometer.
Focal adhesions were visualized in cells fixed for 10 min with 4% formaldehyde in PBS and 0.1% Triton X-100, as described previously (Levy et al., 2000). Cells were examined at RT with a confocal laser scanning microscope (model LSM 510; Carl Zeiss MicroImaging, Inc.) and software (Carl Zeiss MicroImaging, Inc.) using a 40/NA 1.2 objective (Carl Zeiss MicroImaging, Inc.).
Adhesion assays
Vitronectin was purified from human plasma (Cheresh and Spiro, 1987) and coated onto 96-well bacterial plates (Evans et al., 2003). Cells were labeled with 5 µM of CellTracker dye (Molecular Probes), harvested with trypsin/EDTA, and resuspended at 4 x 105 cells/ml in TBS containing 1 mM Mn2+. 100 µl of cell suspension was added per well and incubated at 37°C for 30 min. The number of adherent cells was quantitated as described previously (Evans et al., 2003).
Anchorage-independent growth and suspension culture
Anchorage independent growth in soft agar was evaluated as described previously (Jones et al., 1996). 5 x 103 cells per 35-mm dish in 0.3% agar were cultured for 3 wk, and then stained with 1 mg/ml of nitroblue tetrazilium dye (Sigma-Aldrich). All colonies >140 µm in diameter in each of the three randomly chosen 0.8-cm2 fields were scored under a dissecting microscope.
Cells were suspended in medium supplemented with 3.5 g/200 ml of methyl cellulose (Sigma-Aldrich), essentially as described previously (Levy et al., 2000), except that the concentration of HICE cocktail was one tenth that in the normal culture medium. The following inhibitors were used at the concentrations indicated: z-VAD-fmk (100µM; ICN Biomedicals), z-LIEH-fmk (100µM; Sigma-Aldrich), z-LEHD-fmk (100µM; Sigma-Aldrich), LY294002 (50 µM; Sigma-Aldrich), SB203580 (10 µM; Sigma-Aldrich), and U0126 (10 µM; Promega).
Western blotting
Cells were lysed on ice in a modified RIPA buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, 5 mM EDTA, and 1% Triton X-100, with protease and phosphatase inhibitor cocktails (Sigma-Aldrich; Hobbs and Watt, 2003). Soluble proteins were resolved by SDS-PAGE on 10% Laemmli gels and transferred onto Immobilon PVDF membranes (Millipore). Antibody labeling was performed as described previously using a chemiluminescence kit (Western Lightning; NEN Life Science Products) for detection (Hobbs and Watt, 2003).
![]() |
Acknowledgments |
---|
S.M. Janes was the recipient of a Medical Research Council Clinical Fellowship. This work was supported by Cancer Research UK.
Submitted: 9 December 2003
Accepted: 10 June 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agrez, M., X. Gu, J. Turton, C. Meldrum, J. Niu, T. Antalis, and E.W. Howard. 1999. The vß6 integrin induces gelatinase B secretion in colon cancer cells. Int. J. Cancer. 81:9097.[CrossRef][Medline]
Ahmed, N., J. Niu, D.J. Dorahy, X. Gu, S. Andrews, C.J. Meldrum, R.J. Scott, M.S. Baker, I.G. Macreadie, and M.V. Agrez. 2002. Direct integrin vß6-ERK binding: implications for tumour growth. Oncogene. 21:13701380.[CrossRef][Medline]
Allombert-Blaise, C., S. Tamiji, L. Mortier, H. Fauvel, M. Tual, E. Delaporte, F. Piette, E.M. DeLassale, P. Formstecher, P. Marchetti, and R. Polakowska. 2003. Terminal differentiation of human epidermal keratinocytes involves mitochondria- and caspase-dependent cell death pathway. Cell Death Differ. 10:850852.[CrossRef][Medline]
Bachelder, R.E., M.A. Wendt, N. Fujita, T. Tsuruo, and A.M. Mercurio. 2001. The cleavage of Akt/protein kinase B by death receptor signaling is an important event in detachment-induced apoptosis. J. Biol. Chem. 276:3470234707.
Breuss, J.M., J. Gallo, H.M. DeLisser, I.V. Klimanskaya, H.G. Folkesson, J.F. Pittet, S.L. Nishimura, K. Aldape, D.V. Landers, W. Carpenter, et al. 1995. Expression of the ß6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. J. Cell Sci. 108:22412251.
Cardone, M.H., N. Roy, H.R. Stennicke, G.S. Salvesen, T.F. Franke, E. Stanbridge, S. Frisch, and J.C. Reed. 1998. Regulation of cell death protease caspase-9 by phosphorylation. Science. 282:13181321.
Carroll, J.M., M.R. Romero, and F.M. Watt. 1995. Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis. Cell. 83:957968.[Medline]
Cheresh, D.A., and R.C. Spiro. 1987. Biosynthetic and functional properties of an Arg-Gly-Asp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen, and von Willebrand factor. J. Biol. Chem. 262:1770317711.
Datta, S.R., H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, and M.E. Greenberg. 1997. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 91:231241.[Medline]
Davies, J., J. Warwick, N. Totty, R. Philp, M. Helfrich, and M. Horton. 1989. The osteoclast functional antigen, implicated in the regulation of bone resorption, is biochemically related to the vitronectin receptor. J. Cell Biol. 109:18171826.[Abstract]
Evans, R.D., V.C. Perkins, A. Henry, P.E. Stephens, M.K. Robinson, and F.M. Watt. 2003. A tumor-associated ß1 integrin mutation that abrogates epithelial differentiation control. J. Cell Biol. 160:589596.
Frisch, S.M. 1999a. Evidence for a function of death-receptor-related, death-domain-containing proteins in anoikis. Curr. Biol. 9:10471049.[CrossRef][Medline]
Frisch, S.M. 1999b. Methods for studying anoikis. Methods Mol. Biol. 129:251256.[Medline]
Frisch, S.M., and H. Francis. 1994. Disruption of epithelial cellmatrix interactions induces apoptosis. J. Cell Biol. 124:619626.[Abstract]
Frisch, S.M., K. Vuori, D. Kelaita, and S. Sicks. 1996. A role for Jun-N-terminal kinase in anoikis; suppression by bcl-2 and crmA. J. Cell Biol. 135:13771382.[Abstract]
Gandarillas, A., L.A. Goldsmith, S. Gschmeissner, I.M. Leigh, and F.M. Watt. 1999. Evidence that apoptosis and terminal differentiation of epidermal keratinocytes are distinct processes. Exp. Dermatol. 8:7179.[Medline]
Gervais, F.G., R. Singaraja, S. Xanthoudakis, C.A. Gutekunst, B.R. Leavitt, M. Metzler, A.S. Hackam, J. Tam, J.P. Vaillancourt, V. Houtzager, et al. 2002. Recruitment and activation of caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner Hippi. Nat. Cell Biol. 4:95105.[CrossRef][Medline]
Gilmore, A.P., A.D. Metcalfe, L.H. Romer, and C.H. Streuli. 2000. Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization. J. Cell Biol. 149:431446.
Grossmann, J. 2002. Molecular mechanisms of "detachment-induced apoptosisAnoikis". Apoptosis. 7:247260.[CrossRef][Medline]
Haapasalmi, K., K. Zhang, M. Tonnesen, J. Olerud, D. Sheppard, T. Salo, R. Kramer, R.A. Clark, V.J. Uitto, and H. Larjava. 1996. Keratinocytes in human wounds express vß6 integrin. J. Invest. Dermatol. 106:4248.[Abstract]
Hobbs, R.M., and F.M. Watt. 2003. Regulation of interleukin-1 expression by integrins and epidermal growth factor receptor in keratinocytes from a mouse model of inflammatory skin disease. J. Biol. Chem. 278:1979819807.
Hood, J.D., R. Frausto, W.B. Kiosses, M.A. Schwartz, and D.A. Cheresh. 2003. Differential v integrin-mediated Ras-ERK signaling during two pathways of angiogenesis. J. Cell Biol. 162:933943.
Janes, S.M., T. Ofstad, D.H. Campbell, F.M. Watt, and D.M. Prowse. 2004. Transient activation of FOXN1 in keratinocytes induces a transcriptional programme that promotes terminal differentiation: contrasting roles of FOXN1 and Akt. J. Cell Sci. In press.
Jones, J., M. Sugiyama, P.M. Speight, and F.M. Watt. 1996. Restoration of vß5 integrin expression in neoplastic keratinocytes results in increased capacity for terminal differentiation and suppression of anchorage-independent growth. Oncogene. 12:119126.[Medline]
Jones, J., F.M. Watt, and P.M. Speight. 1997. Changes in the expression of v integrins in oral squamous cell carcinomas. J. Oral Pathol. Med. 26:6368.[Medline]
Jost, M., T.M. Huggett, C. Kari, and U. Rodeck. 2001. Matrix-independent survival of human keratinocytes through an EGF receptor/MAPK-kinase-dependent pathway. Mol. Biol. Cell. 12:15191527.
Kane, L.P., V.S. Shapiro, D. Stokoe, and A. Weiss. 1999. Induction of NF-B by the Akt/PKB kinase. Curr. Biol. 9:601604.[CrossRef][Medline]
Kennedy, S.G., E.S. Kandel, T.K. Cross, and N. Hay. 1999. Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol. Cell. Biol. 19:58005810.
Khwaja, A., and J. Downward. 1997. Lack of correlation between activation of Jun-NH2-terminal kinase and induction of apoptosis after detachment of epithelial cells. J. Cell Biol. 139:10171023.
Kohn, A.D., A. Barthel, K.S. Kovacina, A. Boge, B. Wallach, S.A. Summers, M.J. Birnbaum, P.H. Scott, J.C. Lawrence, Jr., and R.A. Roth. 1998. Construction and characterization of a conditionally active version of the serine/threonine kinase Akt. J. Biol. Chem. 273:1193711943.
Koistinen, P., and J. Heino. 2002. The selective regulation of vß1 integrin expression is based on the hierarchical formation of
v-containing heterodimers. J. Biol. Chem. 277:2483524841.
Legg, J., U.B. Jensen, S. Broad, I. Leigh, and F.M. Watt. 2003. Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis. Development. 130:60496063.
Levy, L., S. Broad, A.J. Zhu, J.M. Carroll, I. Khazaal, B. Peault, and F.M. Watt. 1998. Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice. Gene Ther. 5:913922.[CrossRef][Medline]
Levy, L., S. Broad, D. Diekmann, R.D. Evans, and F.M. Watt. 2000. ß1 integrins regulate keratinocyte adhesion and differentiation by distinct mechanisms. Mol. Biol. Cell. 11:453466.
Lu, Y., Y.Z. Lin, R. LaPushin, B. Cuevas, X. Fang, S.X. Yu, M.A. Davies, H. Khan, T. Furui, M. Mao, et al. 1999. The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells. Oncogene. 18:70347045.[CrossRef][Medline]
Matter, M.L., and E. Ruoslahti. 2001. A signaling pathway from the 5ß1 and
vß3 integrins that elevates bcl-2 transcription. J. Biol. Chem. 276:2775727763.
Mercurio, A.M., and I. Rabinovitz. 2001. Towards a mechanistic understanding of tumor invasionlessons from the 6ß4integrin. Semin. Cancer Biol. 11:129141.[CrossRef][Medline]
Miranti, C.K., and J.S. Brugge. 2002. Sensing the environment: a historical perspective on integrin signal transduction. Nat. Cell Biol. 4:E83E90.[CrossRef][Medline]
Mirza, A.M., A.D. Kohn, R.A. Roth, and M. McMahon. 2000. Oncogenic transformation of cells by a conditionally active form of the protein kinase Akt/PKB. Cell Growth Differ. 11:279292.
Munger, J.S., X. Huang, H. Kawakatsu, M.J. Griffiths, S.L. Dalton, J. Wu, J.F. Pittet, N. Kaminski, C. Garat, M.A. Matthay, et al. 1999. The integrin vß6 binds and activates latent TGF ß1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell. 96:319328.[Medline]
Pasqualini, R., J. Bodorova, S. Ye, and M.E. Hemler. 1993. A study of the structure, function and distribution of ß5 integrins using novel anti-ß5 monoclonal antibodies. J. Cell Sci. 105:101111.
Ramos, D.M., M. But, J. Regezi, B.L. Schmidt, A. Atakilit, D. Dang, D. Ellis, R. Jordan, and X. Li. 2002. Expression of integrin ß6 enhances invasive behavior in oral squamous cell carcinoma. Matrix Biol. 21:297307.[CrossRef][Medline]
Regezi, J.A., D.M. Ramos, R. Pytela, N.P. Dekker, and R.C. Jordan. 2002. Tenascin and ß6 integrin are overexpressed in floor of mouth in situ carcinomas and invasive squamous cell carcinomas. Oral Oncol. 38:332336.[CrossRef][Medline]
Rytömaa, M., L.M. Martins, and J. Downward. 1999. Involvement of FADD and caspase-8 signalling in detachment-induced apoptosis. Curr. Biol. 9:10431046.[CrossRef][Medline]
Rytömaa, M., K. Lehmann, and J. Downward. 2000. Matrix detachment induces caspase-dependent cytochrome c release from mitochondria: inhibition by PKB/Akt but not Raf signalling. Oncogene. 19:44614468.[CrossRef][Medline]
Scatena, M., M. Almeida, M.L. Chaisson, N. Fausto, R.F. Nicosia, C.M. Giachelli, P.D. Arora, J. Ma, W. Min, T. Cruz, and C.A. McCulloch. 1998. NF-B mediates
vß3 integrininduced endothelial cell survival. J. Cell Biol. 141:10831093.
Sonoda, Y., Y. Matsumoto, M. Funakoshi, D. Yamamoto, S.K. Hanks, and T. Kasahara. 2000. Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60. J. Biol. Chem. 275:1630916315.
Stambolic, V., A. Suzuki, J.L. de la Pompa, G.M. Brothers, C. Mirtsos, T. Sasaki, J. Ruland, J.M. Penninger, D.P. Siderovski, and T.W. Mak. 1998. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell. 95:2939.[Medline]
Stupack, D.G., X.S. Puente, S. Boutsaboualoy, C.M. Storgard, and D.A. Cheresh. 2001. Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins. J. Cell Biol. 155:459470.
Sugiyama, M., P.M. Speight, S.S. Prime, and F.M. Watt. 1993. Comparison of integrin expression and terminal differentiation capacity in cell lines derived from oral squamous cell carcinomas. Carcinogenesis. 14:21712176.[Abstract]
Thomas, G.J., M.P. Lewis, I.R. Hart, J.F. Marshall, and P.M. Speight. 2001a. vß6 integrin promotes invasion of squamous carcinoma cells through up-regulation of matrix metalloproteinase-9. Int. J. Cancer. 92:641650.[CrossRef][Medline]
Thomas, G.J., M.P. Lewis, S.A. Whawell, A. Russell, D. Sheppard, I.R. Hart, P.M. Speight, and J.F. Marshall. 2001b. Expression of the vß6 integrin promotes migration and invasion in squamous carcinoma cells. J. Invest. Dermatol. 117:6773.
Toker, A., and A.C. Newton. 2000. Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site. J. Biol. Chem. 275:82718274.
Valentijn, A.J., A.D. Metcalfe, J. Kott, C.H. Streuli, and A.P. Gilmore. 2003. Spatial and temporal changes in Bax subcellular localization during anoikis. J. Cell Biol. 162:599612.
van der Flier, A., and A. Sonnenberg. 2001. Function and interactions of integrins. Cell Tissue Res. 305:285298.[CrossRef][Medline]
Watt, F.M. 2002. Role of integrins in regulating epidermal adhesion, growth and differentiation. EMBO J. 21:39193926.
Related Article