extfieldsofstudy: Biology Medicine

text:
Role of PI 3-kinase, Akt and Bcl-2–related proteins in sustaining the survival of neurotrophic factor–independent adult sympathetic neurons

By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.

Introduction
The molecular mechanisms that regulate neuronal survival and bring about apoptosis have been extensively studied in developing sympathetic neurons. In vivo studies using function-blocking anti-NGF and anti-NT3 antibodies (Levi-Montalcini, 1987;Zhou and Rush, 1995) together with detailed analysis of the timing of neuronal death in the sympathetic chain of mice lacking NGF or NT3 (Crowley et al., 1994;Wyatt et al., 1997;Francis et al., 1999) have shown that sympathetic neurons become dependent on the supply of these two neurotrophins shortly after they begin to innervate their targets. Because NGF and NT3 are normally produced in limiting amounts, only a proportion of the neurons initially generated survive into adulthood, with superfluous neurons being eliminated by apoptosis during the late fetal and early postnatal period. Sympathetic neurons cultured during this period of development die unless NGF or NT3 is added to the culture medium. However, these neurons lose dependence on neurotrophins with age, and, by adulthood, most sympathetic neurons are able to survive for at least a week in culture in defined medium without added neurotrophins (Orike et al., 2000).
Considerable progress has been made in delineating the molecular mechanisms that mediate the survival-enhancing effects of NGF and NT3 on embryonic and neonatal sympathetic neurons. Analysis of the Trk receptor tyrosine kinase isoforms expressed by sympathetic neurons (Wyatt et al., 1997) together with in vitro studies of TrkC-deficient neurons  and the effects of a mutant NT-3 protein that only signals via TrkA (Belliveau et al., 1997) have shown that both NGF and NT-3 promote sympathetic neuron survival by acting via TrkA. Ligand binding causes TrkA dimerization and autophosphorylation on specific tyrosine residues that form docking sites for several adapter proteins including Shc, PLC ␥ , and SHP. These proteins couple Trk to several intracellular signaling pathways that result in Shc/Grb2/Gab1-dependent activation of phospho-inositide (PI)* 3-kinase (Holgado-Madruga et al., 1997) and the production of 3 Ј -phosphorylated phosphoinositides, Shc/Grb2/Sos-dependent activation of Ras, and PLC-␥mediated generation of DAG and inositol trisphosphate .
Studies using function-blocking Ras antibodies or dominant-negative proteins have shown that Ras plays an important role in mediating the survival response of SCG neurons to NGF (Nobes et al., 1996;Markus et al., 1997;Mazzoni et al., 1999). Activated Ras stimulates several downstream signaling cascades including PI 3-kinase and the mitogenactivated protein kinase kinase (MEK)-mitogen-activated protein (MAP) kinase pathways (Shields et al., 2000). Studies using Ras effector mutants suggest that Ras exerts its effects on sympathetic neuron survival principally by activating the catalytic subunit of PI 3-kinase (Rodriguez-Viciana et al., 1994;Mazzoni et al., 1999;Xue et al., 2000), whereas MAP kinase activation plays only a minor role or no role at all (Creedon et al., 1996;Virdee and Tolkovsky, 1996;Klesse and Parada, 1998;Mazzoni et al., 1999;Xue et al., 2000). Although overexpression of P1 3-kinase prevents the death of sympathetic neurons after NGF withdrawal, suggesting that PI 3-kinase is sufficient for survival (Philpott et al., 1997;Crowder and Freeman, 1998), evidence for the involvement of PI 3-kinase in mediating the survival-promoting actions of NGF on embryonic and neonatal sympathetic neurons is conflicting. Whereas several studies have reported that pharmacological or dominant-negative inactivation of PI 3-kinase inhibits the NGF survival response (Crowder and Freeman, 1998;Mazzoni et al., 1999;Vaillant et al., 1999), other studies have reported only modest effects of PI 3-kinase inactivation on the survival of developing sympathetic neurons maintained with NGF (Philpott et al., 1997;Virdee et al., 1999;Tsui-Pierchala et al., 2000). Recently, it has been shown that there is a more critical dependence on PI 3-kinase for survival of sympathetic neurons supported by NGF acting on their distal axons as compared with neurons supported by NGF acting directly on their cell bodies (Kuruvilla et al., 2000).
The serine/threonine protein kinase Akt (protein kinase B), a downstream effector of PI 3-kinase, appears to play a key role in mediating the NGF survival response of developing sympathetic neurons since constitutively active Akt prevents the death of these neurons after NGF deprivation and dominant-negative Akt kills these neurons in the presence of NGF (Crowder and Freeman, 1998;Virdee et al., 1999). Akt influences the activity of several transcription factors implicated in regulating cell survival, including Forkhead 1 , NF-B (Kane et al., 1999;Ozes et al., 1999), and CREB (Du and Montminy, 1998). Phosphorylation of Forkhead 1 by Akt prevents it from inducing expression of the cytotoxic factor FasL whose synthesis after growth factor withdrawal plays a role in causing neuronal apoptosis by initiating a caspase cascade Le-Niculescu et al., 1999;Raoul et al., 1999). Induction of NF-B and CREB transcriptional activity by Akt results in expression of their target genes that include those encoding Bcl-2 and inhibitor of apoptosis proteins, which are required for sympathetic neuron survival in the presence of NGF (Riccio et al., 1999;Wiese et al., 1999). Akt also phosphorylates Bad, a proapoptotic member of the Bcl-2 family, which may prevent it from interacting with and inactivating antiapoptotic members of this family (Datta et al., 1997;del Peso et al., 1997).
Proteins of the Bcl-2 family play a key role in controlling the activation of caspases, which are the proteases that dismantle the cell during apoptosis (Korsmeyer, 1999;Vaux and Korsmeyer, 1999). Bcl-2-related proteins fall into two groups that generally either repress apoptosis (e.g., Bcl-2 and Bcl-x L ) or promote apoptosis (e.g., Bax, Bcl-x s , Bak, and Bad). These proteins influence caspase activation in part by controlling the release of cytochrome c from mitochondria that interacts with the adapter protein Apaf-1, which in turn activates procaspase-9 (Li et al., 1997;Qin et al., 1999). Proapoptotic members like Bax and Bak increase mitochondrial permeability allowing cytochrome c to pass into the cytosol, whereas antiapoptotic members like Bcl-2 and Bcl-x L prevent cytochrome c release (Kharbanda et al., 1997;Kluck et al., 1997;Yang et al., 1997;Shimizu et al., 1999). In addition, Bcl-2 is also able to regulate activation of membrane-associated procaspase-3 independently of cytochrome c (Krebs et al., 1999).
Although a good deal is known about the molecular mechanisms that mediate the survival effects of neurotrophins and induce apoptosis after their withdrawal in developing neurons, the mechanisms that sustain the survival of adult neurotrophic factor-independent neurons has received very little attention. One experimental paradigm used to address the intriguing issue of neurotrophic factor independence has involved growing embryonic sympathetic or sen-*Abbreviations used in this paper: MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase kinase; PI, phosphoinositide; SCG, superior cervical ganglion. sory neurons in culture with NGF until they reach a point when they continue to survive after NGF is withdrawn. In sensory neurons grown under these conditions, inhibition of PI 3-kinase did not compromise survival after NGF withdrawal, suggesting that PI 3-kinase activation is not required for sustaining neuronal survival in this model of neurotrophin independence (Vogelbaum et al., 1998). In embryonic sympathetic neurons grown under these conditions, there is evidence for a block in the apoptotic pathway just upstream of caspase activation close to the point at which Bax acts (Greenlund et al., 1995;Easton et al., 1997). To investigate the molecular mechanisms that underlie neurotrophin independence in adult neurons, we purified and cultured sympathetic neurons from the superior cervical ganglion (SCG) of adult rats. We show that PI-3 kinase/Akt activation and the expression of antiapoptotic members of the Bcl-2 family are required for sustaining the survival of these neurons in the absence of added neurotrophic factors.

Mature sympathetic neurons survive in isolation
Although it has been reported that mature SCG neurons survive without added neurotrophic factors in dissociated cultures enriched for neurons (Orike et al., 2000), it is possible that the small number of nonneuronal cells present in these cultures might produce sufficient neurotrophic factors to sustain neuronal survival. To demonstrate conclusively that mature sympathetic neurons are capable of surviving independently of factors produced by other cells, 12-wk adult rat SCG neurons were plated at single cell density in 96-well plates. The great majority of these neurons ( Ͼ 75%) survived for at least 5 d in defined, serum-free medium without added neurotrophic factors, and their survival was not enhanced by coculturing them with nonneuronal cells in these wells ( Fig.  1). Having established that mature sympathetic neurons can survive autonomously and independently of trophic support from other cells, we embarked upon a series of experiments to elucidate the molecular basis of their survival independence.

PI 3-kinase activation is required for adult SCG neuron survival
We began our investigations of the survival independence of adult SCG neurons using pharmacological agents to inhibit signaling pathways that have been implicated in mediating the survival effects of growth factors. These and all subsequent experiments were carried out in low density-dissociated cultures of SCG neurons ( Ͻ 1,000 neurons per 60-mm petri dish) from which most nonneuronal cells were removed by differential sedimentation ( Ͼ 90% neurons). Addition of Wortmannin or LY294002 at concentrations shown previously to preferentially block PI 3-kinase activation (Okada et al., 1994, Vlahos et al., 1994 caused the rapid death of the majority of the neurons (Fig. 2). Addition of PD98059 to the culture medium at concentrations shown previously to inhibit activation of MEK (Alessi et al., 1995) had little effect on neuronal survival, even when added repeatedly to the medium at daily intervals (Fig. 2). These results suggest that activation of PI 3-kinase is required for the neurotrophic factor-independent survival of adult SCG neurons, whereas MEK/MAP kinase signaling has no apparent role.
To confirm the results of these pharmacological experiments, SCG neurons were injected with a plasmid that expresses ⌬ p85, a dominant-negative form of the regulatory subunit of Class I A PI 3-kinase that is unable to bind to the catalytic p110 subunit, and thereby inhibits endogenous PI 3-kinase activity by sequestering upstream stimulatory proteins that bind to p85 . Whereas neurons injected with an empty expression plasmid survived just as well as uninjected neurons (unpublished data), indicating that the injection procedure is not detrimental for the neurons, injection of the ⌬ p85 expression plasmid caused the death of a substantial number of the neurons (Fig. 3). Although the neurons did not die as fast as neurons treated with PI 3-kinase inhibitors (which may reflect the time taken to synthesize effective levels of ⌬ p85), by 5 d, only 25% of the neurons injected with the ⌬ p85 plasmid were surviving compared with Ͼ 80% of control-injected neurons, suggesting that activation of Class I A PI 3-kinase is required for sustaining the survival of adult sympathetic neurons in defined medium lacking added neurotrophic factors.
Further proof that PI 3-kinase activation is required for the survival of adult SCG neurons came from studying the effects of expressing a recently identified protein termed Ruk l that interacts with the p85 regulatory subunit of Class I A PI 3-kinase and inhibits the lipid kinase activity of the holoenzyme (Gout et al., 2000). Injection of the Ruk l expression plasmid into adult SCG neurons killed a substantial number of these neurons, leaving only 30% surviving 5 d af- ter injection. In contrast, expression of either a shorter Ruk isoform (Ruk m ) that lacks two of the three SH3 domains found in Ruk l (Gout et al., 2000) or a Ruk l deletion mutant (Ruk ⌬ )that lacks the proline-rich and cysteine-rich domains, but retains all three SH3 domains, did not affect the survival of adult SCG neurons. 5 d after injection of the Ruk m or Ruk ⌬ expression plasmids, the number of surviving neurons was no different from control-injected neurons (Fig. 3). The neurons were injected with expression plasmids containing the ⌬p85 dominant-negative form of PI 3-kinase, Ruk l , Ruk m , a Ruk deletion mutant that lacks the proline-rich and cysteine-rich domains (Ruk ⌬ ), or an empty plasmid (control plasmid). The neurons were initially grown for 12 h before injection, and the number of neurons surviving at intervals after injection is expressed as a percentage of the initial number of neurons injected. The means and standard errors for the combined results of three separate experiments are shown.
These latter results are excellent controls for the microinjection experiments and for the Ruk l expression studies as they show that expression of proteins that are closely related to a physiological inhibitor of Class I A PI 3-kinase has no detrimental effect on neuronal survival. Taken together, these results demonstrate that PI 3-kinase activation is required for the survival of adult SCG neurons.

Akt activation is required for adult SCG neuron survival
Because the downstream effector of PI 3-kinase, Akt, plays a key role in mediating the survival response of neurons to neurotrophins (Crowder and Freeman, 1998;Vaillant et al., 1999), we investigated whether Akt activation is required for the survival of adult SCG neurons. Injection of an expression plasmid that directs the synthesis of a kinase-inactive mutant of Akt that acts as a dominant-negative protein (Virdee et al., 1999) caused the death of a substantial number of adult SCG neurons. Over 70% of these neurons had died 5 d after injection with the dominant-negative Akt expression plasmid compared with 20% of control-injected neurons (Fig. 4). The magnitude of neuronal death effected by expression of dominant-negative Akt was similar to that caused by expression of ⌬ p85 or overexpression of Ruk l . These results suggest that Akt is the main downstream effector of PI 3-kinase in sustaining the survival of adult SCG neurons in the absence of added neurotrophic factors.
The demonstration that Akt plays a major role in mediating the PI 3-kinase survival signal in adult sympathetic neurons enabled us to carry out an additional control for the specificity of the pharmacological inhibition of PI 3-kinase by LY294002. Microinjecting a plasmid that expresses a constitutively active Akt protein (see Burgering and Coffer, 1995 for a description of the Gag-PKB protein) was able to rescue 73.2% of the neurons that would have otherwise died after 48-h incubation with 20 M LY294002. The failure to rescue all neurons could be because Akt is not expressed at an optimal level.

Phospho-Akt is present in adult SCG neurons surviving without neurotrophic factors
To ascertain if the active, phosphorylated form of Akt is present in adult SCG neurons surviving without neurotrophic factors and whether this is dephosphorylated after inhibition of PI 3-kinase, we used immunocytochemistry to visualize Akt protein and phospho-Akt protein in SCG neurons grown with and without the PI 3-kinase inhibitor LY294002. Fig. 5 A shows that SCG neurons were clearly immunoreactive for phospho-Akt protein when grown at low density in defined medium alone. Although phospho-Akt immunoreactivity was clearly reduced by treatment with LY294002 for 45 min (Fig. 5G), expression of total Akt protein was little affected as revealed by the similar level of Akt immunofluorescence in the presence and absence of LY294002 (Fig. 5, B and C). There was no obvious change in the expression of either phospho-Akt immunoreactivity or total Akt protein in adult SCG neurons treated for 45 min with NGF (Fig. 5, A and E). No staining was observed when the primary antibodies were omitted from the staining protocol (Fig. 5 H), indicating that there was no detectable nonspecific staining. Taken together, these results suggest  that Akt is constitutively active in adult SCG neurons surviving without neurotrophic factors and that PI 3-kinase is required for sustaining Akt activation.

Adult SCG neurons die by a caspase-dependent apoptotic mechanism after PI 3-kinase inhibition
Adult sympathetic neurons surviving without neurotrophic factors had smooth cell bodies, a smooth spherical nucleus, and unbroken short neurites (Fig. 6 A). Neurons grown without neurotrophins retained this appearance throughout the period of the study (up to 5 d in vitro). However, neurite outgrowth could be stimulated from these neurons after this prolonged period of culture without neurotrophins by adding NGF to the culture medium (unpublished data). This indicates that although NGF is not required for survival, it is capable of promoting neurite outgrowth from these neurons. A similar phenomenon has been observed for neonatal Bax-deficient sympathetic neurons that survive in culture without NGF, but display very poor neurite outgrowth unless NGF is added to the medium (Deckwerth et al., 1996).
Neurons dying as a consequence of LY294002 treatment exhibited cell body condensation, neurite fragmentation, and chromatin condensation (Fig. 6, B and D) that is typical of neuronal apoptosis (Allsopp et al., 1993). To investigate whether caspase activation is required for neuronal death after LY294002 treatment, the caspase inhibitor Z-VAD-FMK (Fearnhead et al., 1995) was added to the culture me-dium to see if this would interfere with the cytotoxic effect of LY294002 and Wortmannin. Whereas Z-VAD-FMK did not affect the survival of neurons growing in defined medium alone (unpublished data), it largely prevented the cell death caused by LY294002 and Wortmannin (Fig. 7), suggesting that adult SCG neurons die by a caspase-dependent mechanism after PI 3-kinase inhibition.

Bcl-x L and Bcl-2 expression is required for adult SCG neuron survival
Because members of the Bcl-2 family of proteins play key roles in controlling caspase activation, we carried out additional experiments to assess the importance of different members of this family in regulating adult sympathetic neuron survival. To ascertain the importance of antiapoptotic members of the Bcl-2 family in sustaining the survival of adult SCG neurons, we injected plasmids expressing specific antisense RNAs to interfere with the synthesis of these proteins. We also injected separate sets of neurons with an empty expression plasmid to control for the injection procedure and studied the effect of overexpressing antiapoptotic proteins. There was no significant difference in the survival of control-injected neurons compared with uninjected neurons (unpublished data), indicating that the microinjection procedure did not affect neuronal survival. Overexpression of the antiapoptotic proteins Bcl-x L or Bcl-2 had little effect on survival. Whereas neurons overexpressing Bcl-x L survived  slightly better than control-injected neurons, the survival of Bcl-2-overexpressing neurons was not significantly different from control-injected neurons (Fig. 8). In contrast, injection of plasmids containing Bcl-x L or Bcl-2 in the reverse orientation caused a marked reduction in neuronal survival. Expression of antisense Bcl-x L had the most dramatic effect, with over three quarters of the neurons dying within the first 24 h after injection, and by 5 d, Ͻ 10% of the neurons were surviving. Expression of antisense Bcl-2 reduced the number of surviving neurons to ف 25% after 5 d (Fig. 8). To confirm that adult SCG neurons normally express Bcl-xL protein and that expression of this protein is reduced by antisense Bcl-xL RNA, we used immunocytochemistry to study Bcl-x L expression after injection of the antisense Bcl-x L plasmid. Whereas control-injected neurons exhibited intense Bcl-x L immunofluorescence (Fig. 6 E), neurons injected with the antisense Bcl-x L plasmid were either unlabeled by the Bcl-x L antiserum or exhibited negligible Bcl-x L immunofluorescence 12 h after injection (Fig. 6 F). Taken together, these results suggest that the endogenous expression of Bcl-x L and, to a lesser, extent Bcl-2 is required for the survival of adult SCG neurons.
In addition to using antisense Bcl-x L expression plasmid to interfere with the endogenous synthesis of the Bcl-x L protein, we also microinjected a plasmid that expresses a proapoptotic isoform of Bcl-x, Bcl-x S which lacks two of the four BH domains present in Bcl-x L (Boise et al., 1993). Expression of Bclx S also caused the rapid death of a large number of SCG neurons, leaving only 20% surviving 5 d after injection (Fig. 8).

Overexpression of Bad or Bax kills adult SCG neurons
In addition to interfering with the synthesis of antiapoptotic members of the Bcl-2 family to ascertain the importance of these proteins in sustaining the survival of adult SCG neurons, we also overexpressed proapoptotic members of this family to see if this would compromise the survival of these neurons. The most marked effect on survival was observed by overexpressing Bad. Injection of a Bad expression plasmid caused the rapid death of the majority of adult SCG neurons, leaving Ͻ 20% surviving 5 d after injection (Fig. 9). Injection of a Bax expression plasmid caused much less neuronal death, leaving 40% surviving 5 d after injection (Fig. 9).

Discussion
We have shown that adult sympathetic neurons are able to survive for an extended period in culture in isolation without the need for trophic support from any other cells. Several complementary experimental observations have clearly demonstrated that the survival of these neurons depends on PI 3-kinase activity and on activation of the downstream effector of PI 3-kinase, the serine/threonine protein kinase Akt. These neurons are killed by pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin, by the expression of a dominant-negative Class I A PI 3-kinase p85 regulatory subunit, by a dominant-negative Akt protein, or by overexpressing Ruk l , which is a natural inhibitor of Class I A PI 3-kinase. These neurons do not die as a nonspecific consequence of plasmid microinjection or the expression of new protein because overexpressing proteins that are very similar to Ruk l (Ruk m and a Ruk l deletion mutant that retains all three SH3 domains) is not detrimental. Furthermore, phospho-Akt staining suggests that Akt is constitutively active in these neurons.
Although the role of PI 3-kinase in mediating the survivalpromoting action of NGF on embryonic and neonatal sympathetic neurons is controversial (Philpott et al., 1997;Crowder and Freeman, 1998;Mazzoni et al., 1999;Vaillant et al., 1999;Virdee et al., 1999;Tsui-Pierchala et al., 2000), studies of several other kinds of neurons and many other cell types have shown that PI 3-kinase and serine/threonine kinase Akt/PKB play major roles in mediating the survival response of many different kinds of cells to a variety of growth factors (Datta et al., 1999). However, our study provides the first evidence that activation of PI 3-kinase and Akt is required to prevent the death of cells that survive independently of extracellular growth factors.
Even though adult SCG neurons survive as single cells in serum-free medium, nonetheless, it is possible that they could be sustained by neurotrophic factors that they produce themselves. Autocrine action of neurotrophic factors has been described previously for BDNF acting on embryonic and adult sensory neurons (Wright et al., 1992;Acheson et al., 1995) and for HGF acting on sympathetic neuroblasts (Maina et al., 1998). Although autocrine trophic support of adult sympathetic neurons is entirely possible, preliminary data suggest that several of the likely candidates are unlikely to be involved. Adult SCG neurons are not killed by the inhibitor of Trk receptor tyrosine kinases K252a (Orike et al., 2000) or by function-blocking antibodies to GFR␣-3, a key component of the artemin receptor (Andres et al., 2001) or HGF (N. Orike, J. Thompson, and A.M. Davies, unpub-lished data). These findings suggest that neither NGF and NT-3, which promote the survival of developing sympathetic neurons (Levi-Montalcini, 1987;Crowley et al., 1994;Zhou and Rush, 1995;Wyatt et al., 1997;Francis et al., 1999), HGF, which enhances the survival of sympathetic neuroblasts (Maina et al., 1998), nor artemin, which is important for the survival of a SCG neurons during the postnatal period (Nishino et al., 1999;Andres et al., 2001), are required for the survival of adult SCG neurons. Furthermore, the activation of PI 3-kinase by NGF in SCG neurons is thought to occur predominantly by direct activation of the p110 catalytic subunit by Ras (Rodriguez-Viciana et al., 1994;Nobes et al., 1996;Markus et al., 1997;Xue et al., 2000). Yet we have shown that inhibiting the function of the p85 regulatory subunit of Class I A PI 3-kinase by expressing a dominant-negative protein or overexpressing Ruk l kills adult SCG neurons, indicating that this regulatory subunit is crucial for maintaining the activity of the catalytic subunit at the level required to sustain the survival of adult SCG neurons.
Our demonstration that PI 3-kinase activity is required for the survival of adult rat sympathetic neurons in the absence of neurotrophic factors differs from the findings of studies of embryonic sympathetic or sensory neurons that attained neurotrophic factor independence in culture after prolonged incubation with NGF. Dorsal root ganglion neurons from E15 rat embryos that were maintained for 3 wk in the presence of NGF survived after NGF withdrawal and were not killed by inhibiting PI 3-kinase activation with LY294002, but were killed by LY294002 treatment at earlier time points before the acquisition of NGF independence (Vogelbaum et al., 1998). E21 rat SCG neurons maintained for 3 wk with NGF also survived after NGF deprivation, but exhibited a rapid decrease in MAP kinase activation and early changes in gene expression after NGF deprivation that are typical of NGF withdrawal before the acquisition of NGF independence. However, like Bax-deficient embryonic neurons that survive after NGF deprivation, embryonic SCG neurons maintained in long-term culture with NGF do not exhibit c-fos induction after NGF deprivation. These observations, together with the finding that overexpression of Bax in these neurons restored their survival dependence on NGF, suggest that the apoptotic pathway was blocked at a point just upstream of caspase activation close to the point at which Bax acts (Greenlund et al., 1995;Easton et al., 1997). Thus, although embryonic sensory and sympathetic neurons lose dependence on NGF after being cultured with this factor for several weeks, the mechanism of neurotrophin independence appears to be different from that of adult sympathetic neurons that rely on PI 3-kinase activity for survival. However, one potential caveat of our study is that we have studied the role of PI 3-kinase shortly after plating when a stress response to axotomy might make them more susceptible to PI 3-kinase inhibition. In future work, it will be important to address the relevance of our in vitro observations to the role of PI 3-kinase in sustaining the survival of adult sympathetic neurons in vivo.
Another signaling pathway that has been implicated in neuronal survival under certain circumstances is the MEK/ MAP kinase pathway. Although this Ras-activated pathway plays either no role or only a minor role in mediating the survival response of developing neurons to NGF (Creedon et al., 1996;Virdee and Tolkovsky, 1996;Klesse and Parada, 1998;Mazzoni et al., 1999;Xue et al., 2000), it plays a major role in BDNF neuroprotection against camptothecin (Hetman et al., 1999) and is capable of reducing p53-dependent apoptosis induced by cytosine arabinoside in sympathetic neurons (Anderson and Tolkovsky, 1999). Our demonstration that the MEK inhibitor PD98059 does not kill adult SCG neurons suggests that the MEK/MAP kinase activation is not required for sustaining the survival of these neurons.
Considerable evidence indicates that members of the Bcl-2 play a key role in regulating neuronal survival during development. Overexpression of Bcl-2 or Bcl-x L promote the survival of developing neurons in vitro and in vivo (Garcia et al., 1992;Allsopp et al., 1993;Martinou et al., 1994;Farlie et al., 1995;Gonzalez-Garcia et al., 1995;Middleton et al., 1996;Parsadanian et al., 1998), whereas reduction or elimination of endogenous Bcl-2 or Bcl-x L reduces neuronal survival (Allsopp et al., 1995;Motoyama et al., 1995;Michaelidis et al., 1996;Piñón et al., 1997). In contrast, Bax overexpression accelerates neuronal death after neurotrophin withdrawal (Vekrellis et al., 1997;Martinou et al., 1998), and reduction or elimination of endogenous Bax promotes the survival of neurons without neurotrophic factors in culture and prevents their death in vivo (Deckwerth et al., 1996;Gillardon et al., 1996;Miller et al., 1997;White et al., 1998;Bar-Peled et al., 1999). Our studies of manipulating the expression of Bcl-2 family proteins in adult sympathetic neurons have likewise shown that these proteins also play an important role in regulating the survival of mature sympathetic neurons. Expression of the proapoptotic proteins Bad, Bax, and Bcl-x S killed these neurons as did anti-sense RNA to the antiapoptotic proteins Bcl-x L and Bcl-2. A link between PI 3-kinase/Akt signaling and the function of these proteins has come from the demonstration that activated Akt phosphorylates Bad, resulting in its sequestration by cytosolic 14-3-3 proteins (Datta et al., 1997;del Peso et al., 1997). Because Bad brings about apoptosis by binding to and inhibiting the antiapoptotic actions of Bcl-x L , the sequestration of Bad in the cytosol by 14-3-3 proteins results in enhanced survival.
In summary, we have shown that the survival of adult sympathetic neurons is dependent on PI 3-kinase/Akt signaling and the expression of the antiapoptotic proteins Bclx L and Bcl-2. In future work, it will be important to ascertain which proteins interact with the p85 regulatory subunit of Class I A PI 3-kinase in adult sympathetic neurons to maintain the activity of the holoenzyme and understand what brings about the change in PI 3-kinase regulation between postnatal animals and the adult.

Culture and microinjection of sympathetic neurons
Dissociated cultures of purified SCG neurons from 12-wk-old Sprague-Dawley rats were set were grown in defined, serum-free medium (Orike et al., 2000). In brief, SCG were cut into small pieces and incubated at 37ЊC for 30 min with 400 IU/ml collagenase class 2 (C9891; Sigma-Aldrich) followed by 40 min with 1 mg/ml trypsin (T2271; Sigma-Aldrich). The enzymes were dissolved in HBSS containing 6 mg/ml BSA (A4716; Sigma-Aldrich). The ganglion fragments were dissociated by gentle trituration using a firepolished glass pipette, and the neurons were purified free of nonneuronal cells by differential sedimentation (Davies, 1986). For microinjection studies, the neurons (Ͼ90% pure) were plated in polyornithine/laminin-coated 60-mm-diam tissue culture petri dishes  at a density of 500 to 1,000 neurons per dish. For single cell cultures, the neurons were plated in 96-well dishes. The cultures were incubated at 37ЊC in a humidified 4.5% CO 2 incubator in defined medium consisting of Ham's F14 supplemented with 2 mM glutamine, 0.35% BSA (Pathocyte-4; ICN Biomedicals), 60 ng/ml progesterone, 16 g/ml putrescine, 400 ng/ml L-thyroxine, 38 ng/ml sodium selenite, and 340 ng/ml triiodothyronine.
The neurons were initially grown overnight and were microinjected intranuclearly with the aid of a manually operated Narishige micromanipulator (Allsopp et al., 1993) with expression plasmids encoding wild-type or mutated signaling proteins or members of the Bcl-2 family in the sense or antisense orientation. Expression plasmids without a cDNA insert were used to control for any nonspecific effects of the injection procedure. The expression plasmids were diluted in 100 mM potassium phosphate buffer, pH 7, to a concentration of 100 ng/ml and filtered through a 0.22-m filter before injection. All neurons within a specific grid square marked on the inside of the culture dish were pressure-injected. Intranuclear injection resulted in Ͼ95% viability assessed 1 h after injection. The number of surviving neurons was counted at 12 or 24 hourly intervals after injection, and is expressed as a percentage of the number injected in the grid square. About 150 neurons were injected for each experimental condition and followed longitudinally in each experiment.
Live and dead neurons were clearly distinguished by the morphology when examined by phase-contrast microscopy. Live neurons had large, smooth, intact cell bodies and smooth, unbroken neurites. Dead neurons rapidly disintegrated, leaving cell body ghosts comprised of cellular debris and fragmented neurites, which are appearances that are typical of neuronal apoptosis in culture (Allsopp et al., 1993). In addition to these characteristic appearances, we also used DAPI to stain nuclear chromatin to determine if the chromatin condensation, which is typical of apoptosis, occurs in adult sympathetic neurons dying as a consequence of inhibiting PI-3 kinase.

Plasmids and pharmacological reagents
A variety of expression plasmids were used to express wild-type or mutated proteins or antisense RNA in cultured sympathetic neurons. cDNAs encoding members of the Bcl-2 family (Bcl-2, Bcl-x L , Bcl-x S , Bad, and Bax) were cloned into the pSFFV vector in the sense and antisense orientations. The pCMV vector was used to express the kinase-inactive mutant of PKB/Akt, Ruk l and Ruk m , and the pSG5 vector was used to express the ⌬p85 dominant-negative form of PI 3-kinase. The PI 3-kinase inhibitors LY294002 and Wortmannin, the MEK inhibitor PD98059, and the caspase inhibitor Z-VAD-FMK were obtained from Calbiochem.

Immunocytochemistry
Cultures of adult SCG neurons were fixed with fresh 4% paraformaldehyde for 15 min, washed in TBS with .1% Triton X-100 (TBT) and blocked with TBT plus 10% goat serum. They were incubated with primary antibody (rabbit polyclonals from Santa Cruz Biotechnology, Inc. and New England Biolabs, Inc.) at a dilution of 1:100 overnight at 4ЊC, washed in TBT, and incubated with FITC-labeled goat anti-rabbit antiserum (Sigma-Aldrich) at a dilution of 1:500 at room temperature for 1 h. The cultures were washed, mounted beneath coverslips, and viewed with an Axioskop fluorescence microscope.


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Rac1 mediates type I collagen-dependent MMP-2 activation. role in cell invasion across collagen barrier.

Cell migration and proteolysis are two essential processes during tumor invasion and metastasis. Matrix metalloproteinase (MMP)-2 (type IV collagenase; gelatinase A), is implicated in tumor metastasis as well as in primary tumor growth. The Rho family of small GTPases regulates the dynamics of actin cytoskeleton associated with cell motility. In this report, we provide evidence that Rac1, one member of Rho-related small GTPases, is a mediator of MMP-2 activation in HT1080 fibrosarcoma cells cultured in three-dimensional collagen gel (3D-col) and that MMP-2 activation is required for Rac1-promoted cell invasion through collagen barrier. Stable expression of dominant negative (Rac1V12N17) and constitutively active Rac1 (Rac1V12), respectively, in HT1080 cells demonstrates that Rac1 promoted cell invasiveness across type I collagen and collagen-dependent MMP-2 activation. Active Rac1 is sufficient to induce MMP-2 activation in cells cultured in fibrin gel, an extracellular matrix component that does not support MMP-2 activation. The Rac1-dependent MMP-2 activation occurred in a cell-associated fashion and required MMP activities. Because the cell membrane-mediated MMP-2 activation requires MT1-MMP and low amount of issue inhibitor of matrix metalloproteinase-2 (TIMP-2), their expression was examined. Rac1 modulated MT1-MMP mRNA level and the accumulation of a 43-kDa form of MT1-MMP protein, in correlation with MMP-2 activation profile. However, TIMP-2 expression was independent of Rac1 activity. The coordinate modulation of MMP-2 activity and MT1-MMP expression/processing by Rac1 is consistent with cell collagenolytic activity. The C-terminal hemopexin-like domain of MMP-2, which interferes with the cell membrane activation of MMP-2, reduced Rac1-promoted cell invasiveness as monitored by collagen invasion assay. These results suggest that collagen-dependent MMP-2 activation and MT1-MMP expression/processing contribute to Rac-promoted tumor cell invasion through interstitial collagen barrier.

During metastasis, invasive cells must traverse tissue barriers comprised largely of type I collagen. This process depends on the ability of tumor cells to degrade the surrounding collagen matrix and then migrate through the matrix defects (1)(2)(3). The actin dynamics regulated by Rho family of small GTPases play a critical role in cell migration (4). The initiation of cell migration is characterized by actin polymerization at the leading edge and extension of a lamella in the direction of motion. Rac1, a member of the Rho family proteins that regulates the assembly of a meshwork of actin filaments at the cell periphery to produce lamellipodia (5), has been implicated in oncogenic transformation (6 -8) and metastasis induction (9). Recent studies have demonstrated the direct role of Rac1 activity in cell invasiveness in type I collagen matrix (10,11). Transfection of non-invasive mammalian epithelial cells T47D with active Rac1 induces cell invasion through type I collagen (10). Similarly, overexpression of adaptor proteins p130 Crk -associated substrate (CAS) 1 /c-CrkII (Crk) induces Rac1-dependent COS-7 cell invasiveness in three-dimensional collagen (3D-col) culture (11). However, the proteolytic activities during Rac1-promoted cell invasion through type I collagen barriers remain undefined.
Increasing evidence has suggested that proteolytic activities at cell surface promote cell invasion (12). Matrix metalloproteinase (MMP)-2 (type IV collagenase; gelatinase A) is a cell surface-associated type I collagen-degrading MMP (13,14). Overexpressed in different types of tumor (15), MMP-2 is involved in tumor metastasis, primary tumor growth, and angiogenesis (2, 16 -19). Although considerable attention has been focused on the role of MMP-2 as a type IV collagenase in cell invasion across basement membrane, recent evidence has implicated MMP-2 in type I collagen remodeling by tumor cells (20) and the tubular organization of endothelial cells in 3D-col (21). In common with all MMPs, MMP-2 is synthesized and secreted as a latent precursor, requiring proteolytic removal of the propeptide for activation. The physiological mechanism that accounts for MMP-2 activation is under intense study. It is suggested that MMP-2 is activated in a membrane-associated mechanism after a two-step process that involves an initial cleavage of the zymogen followed by an autocatalytic conversion of the intermediate into a fully active enzyme (22)(23)(24)(25). MT1-MMP, the best characterized member of MT-MMPs, is believed to carry out the initial cleavage of MMP-2 after the binding of MMP-2 proenzyme to an MT1-MMP⅐TIMP-2 complex at the cell surface (23,26,27). Interestingly, MT1-MMP becomes degraded to an inactive 43-kDa form during the MMP-2 activation (28 -31). MT1-MMP is also overexpressed in several tumor tissues where activated MMP-2 is found (19). In addition to its ability to activate MMP-2, MT1-MMP has intrinsic ECM degrading activity (32)(33)(34)(35)(36). Therefore MT1-MMP and MMP-2 activities at the cell surface provide a powerful combination for the localized ECM remodeling (33,37).
To evaluate the hypothesis that MT1-MMP/MMP-2 proteolytic cascade might play a functional role in Rac1-induced tumor cell invasion through type I collagen-rich tissue barrier, we examined an invasive HT1080 fibrosarcoma cell line that showed elevated level of active MMP-2 during cell-fibrillar collagen interaction (38). In this report, we provide evidence that Rac1 is a mediator of collagen-stimulated MMP-2 activation and MT1-MMP expression/processing, collagenolytic activity, and cell invasion through 3D-col. Furthermore, active MMP-2 contributes to Rac1-induced collagen invasive activity. Our findings suggest that Rac1 mediates MMP-2 activation and MT1-MMP expression/processing during the encounter between invading tumor cells and type I collagen-rich stroma, thereby facilitating collagenolysis and cell invasion.

MATERIALS AND METHODS
Cell Culture and Plasmids-HT1080 fibrosarcoma and HEp3 epidermoid carcinoma cell lines were maintained in DMEM containing 10% heat-inactivated fetal bovine serum (Hyclone) supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin and streptomycin (Life Technologies, Gaithersburg, MD). Stably transfected cell lines were maintained in medium that included 200 g/ml G418 in addition to the above mentioned supplements. Plasmids RSV-neo-␤galactosidase, c-Myc-tagged Rac1V12N17, and Rac1V12 were kindly provided by Dr. Lorne Taichman, SUNY at Stony Brook, and Dr. Alan Hall, University College London, London, United Kingdom. Plasmids containing cDNAs for MT1-MMP, MMP-2, TIMP-2, and 36B4 were purchased from ATCC.
Stable Transfection of HT1080 Cell Line-Subconfluent cell culture was co-transfected with one of testing plasmids (Rac1V12N17, Rac1V12, and vector) and a plasmid construct containing the neomycinresistant gene by the calcium phosphate precipitation technique as previously described (39). After transfection, individual colonies from Rac1V12N17-and Rac1V12-transfected cells were isolated after 2-3 weeks of 500 g/ml G418 selection. As control, vector-transfected clones were pooled. All stably transfected cell lines were maintained in growth medium containing 200 g/ml G418.
Treatment of Cells with Concanavalin A-Cells grown on tissue culture plates in growth medium (10% heat-inactivated fetal bovine serum/DMEM) were washed twice with serum-free medium before the addition of serum-free medium supplemented with 0.2% heat-inactivated lactoalbumin and 50 g/ml concanavalin A (Sigma). Medium was then collected after 16 -24 h for zymography.
Three-dimensional Collagen and Fibrin Cell Culture-Type I collagen or fibrin gel cultures were prepared according to a procedure previously described with some modification (40). Pepsin-solubilized bovine dermal collagen dissolved in 0.012 M HCl was 99.9% pure containing 95-98% type I collagen and 2-5% type III collagen (Vitrogen 100, Collagen Corp). Briefly, cells detached from tissue culture plates in growth medium were washed with warm DMEM twice before being seeded into a serum-free solution that contained 2 mg/ml vitrogen or 3 mg/ml fibrinogen (Calbiochem-Novabiochem). Subsequently, cell-collagen or cell-fibrinogen suspension (5 ϫ 10 5 cells/ml) was plated onto 24-well plates at 250 l/well or 35-mm plastic dishes at 1.5 ml/dish. Cell-collagen cultures were incubated at 37°C to form gel. The gelling of cell-fibrin cultures occurred in less than 5 min at room temperature after the addition of thrombin to a final concentration of 0.2 unit/ml. After both collagen and fibrin cell cultures formed gel, serum-free DMEM supplemented with 0.2% heat-inactivated lactoalbumin was added. Gels remained attached to the plastic dish for the duration of incubation.
Substrate Zymography-Conditioned medium was collected from cells cultured for 18 -24 h in serum-free medium. To analyze the activity of cell-associated MMP-2, cells (7.5 ϫ 10 5 ) in 35-mm plates were released from collagen or fibrin gel after digestion with bacterial collagenase D (Roche Molecular Biochemicals, Indianapolis, IN) for 10 min or dispase (Becton Dickinson, Bedford, MA) for 5 min, respectively. Cells were washed gently with ice-cold phosphate-buffered saline 10 -12 times and suspended in 150 l of 1 ϫ SDS sample buffer. Aliquots (total cell lysates) were immediately processed for enzymatic assay using zymography. Substrate zymography was performed as described previ-ously with some modifications (41,42). SDS-polyacrylamide (12% unless indicated in the figure legend) gels were co-polymerized with 1 mg/ml gelatin or 0.5 mg/ml type I collagen (Sigma). Samples (conditioned medium or total cell lysates) were resolved under nonreducing conditions. Gels were washed twice in 2.5% Triton X-100 for 30 min and incubated overnight in a buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM CaCl 2 , and 0.02% NaN 3 (gelatin substrate) or 100 mM Tris-HCl, pH 8.0, 5 mM CaCl 2 , 0.005% Brij-35, and 0.02% NaN 3 (type I collagen substrate). At the end of the incubation, gels were stained with Coomassie Blue and destained.
Staining of Actin Cytoskeleton-Actin organization was visualized by staining with Texas Red-conjugated phalloidin (Molecular Probes, Eugene, OR). Cells in serum-free vitrogen solution were plated in 8-well chamber slides to form gel. After 24 h, cells in collagen gel were fixed in 4% paraformaldehyde, permeabilized with acetone at Ϫ20°C, and stained with Texas Red-conjugated phalloidin for 30 min at room temperature. The images were captured using an epifluorescence microscope by the University Microscopy Imaging Center, SUNY at Stony Brook.
Northern Blot Analysis-Total RNA was isolated from cells cultured in collagen or fibrin gel for 10 h and Northern analysis was performed as previously described (40). RNA was detected with ␣-32 P-labeled cDNA probes for MT1-MMP and TIMP-2. Control probe was 36B4 cDNA.
Western Blot Analysis-Cell extracts were prepared using a detergent extraction method described by Lee et al. (30) with a few modifications. Briefly, cells cultured in collagen or fibrin gel were washed gently with cold phosphate-buffered saline three times, transferred into a 1.5-ml microcentrifuge tube, and subject to centrifugation to remove as much solution as possible. The pellet was suspended in a Triton X-100 lysis buffer (2% Triton X-100 in 100 mM Tris buffer, pH 7.5, and 150 mM NaCl in the presence of protein inhibitors: 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin) and forced through a syringe with a 26-gauge needle several times. The supernatant after centrifugation was designated as cell extracts for Western analysis. Western blotting analysis was performed as previously described (43). After detection by monoclonal antibodies against Rac1 (Upstate Biotechnology, Lake Placid, NY), MMP-2 (Chemicon, Temecula, CA), TIMP-2, and MT1-MMP (Calbiochem-Novabiochem), the blots were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).
Collagen Fibril Dissolution-The capacity of cells to degrade type I collagen fibrils was assessed based on the modification of a procedure as described (35). Briefly, 300 l of vitrogen solution at 1 mg/ml was added to each well of a 24-well plate and allowed to air-dry overnight. The collagen fibril film was washed with several changes of distilled water and serum-free DMEM before a pellet of 4 ϫ 10 4 cells in 25 l of 10% fetal bovine serum/DMEM was dotted onto the center of each well. Cells were allowed to attach for 5 h, washed twice with serum-free DMEM, and then incubated for 3 days in serum-free DMEM supplemented with 0.2% lactoalbumin. Following the incubation, cells were removed with trypsin/EDTA and collagen remaining in the wells was visualized by staining with Coomassie Blue.
For quantification of fibrillar type I collagen degradation, type I rat tail collagen (Becton Dickinson) was labeled with [ 3 H]acetic anhydride (Amersham Pharmacia Biotech) according to the protocols described (44,45). 150 l of 3 H-labeled type I collagen (0.5-1 ϫ 10 6 cpm/mg protein) was allowed to polymerize in individual wells of a 48-well plate. Cells at 1.5 ϫ 10 4 /300 l were added to each well and incubated at 37°C. To follow the progressive degradation of collagen fibrils, aliquots (50 l) of the medium were collected after 24 and 48 h. The soluble radioactivity was quantified in a liquid scintillation counter.
Expression and Preparation of MMP-2 Domains-A cDNA clone K-121 that contains the coding sequence for MMP-2 with a partially truncated propeptide domain (46) was used as a template for amplification with polymerase chain reaction. The regions corresponding to residues Gly 417 -Cys 631 (C-terminal hemopexin-like domain; CTD) or Val 191 -Gln 364 (fibronectin type-II-like modules; CBD) of MMP-2 proenzyme, respectively, were amplified. The resulting fragments were inserted into a pGEX-3X plasmid, respectively (Amersham Pharmacia Biotech, Uppsala, Sweden). The glutathione S-transferase fusion proteins were purified on Sepharose 4B-coupled glutathione beads (Amersham Pharmacia Biotech) based on the manufacturer's instructions.
Collagen Invasion Assay-The assay was performed using the modification of procedures previously described (47). 50 l of vitrogen solution at 1 mg/ml was applied to the upper compartment of each well in a 24-well Transwell plate (8-m pore size; Costar) and allowed to gel at 37°C. Cells at 5 ϫ 10 4 in 200 l of serum-free DMEM were added to the upper chamber. Culture medium was added to the lower compartment. In indicated experiments, inhibitors or controls were added to both upper and lower chambers at the following final concentrations: 25 M SC68180 (formerly SC44463, N-[3-(NЈ-hydroxycarboxamido)-2-(2-methylpropyl)propanoyl]-O-methyl-L-tyrosine-N-methylamide; a generous gift of Dr. W. C. Parks, Washington University, St. Louis), 100 g/ml aprotinin (Sigma), 500 ng/ml recombinant TIMP-1 (Chemicon), 500 ng/ml recombinant TIMP-2 (Chemicon), 100 M furin inhibitor Dec-Arg-Val-Lys-Arg-CH 2 Cl (Bachem Biochemicals), 500 ng/ml CTD (Gly 417 -Cys 631 ), and 500 ng/ml CBD (Val 191 -Gln 364 ). The invasion proceeded for 24 h at 37°C. After incubation, the filters were fixed and stained with Diff-Quick staining kit (Fisher Scientific). The cells that reached the underside of the filter were counted. For each filter, the number of cells in 10 randomly chosen microscope fields was determined and averaged. Three invasion chambers were used per condition. The final values were the average of triplicates.

HT1080 Cells Stably Expressing Rac1 Mutants Exhibit Distinct Morphology and Invasive Properties in Three-dimensional
Collagen Gel-A human fibrosarcoma cell line, HT1080, was transfected with dominant negative Rac1V12N17, constitutively active Rac1V12, or a vector control. Ectopic Rac1 expression was examined by Western analysis in G418-resistant clones. In a representative experiment, a monoclonal antibody against Rac1 detected both ectopic Rac1 tagged with c-Myc (upper band) and endogenous Rac1 (lower band) in lysates of cells expressing Rac1V12N17 (HN) and Rac1V12 (HV), but only endogenous Rac1 in vector-transfected cell extracts (HW) (Fig. 1A). Clones that express comparable levels of Rac1 were selected for further study.
Cells were embedded in 3D-col in serum-free medium for 24 h and subject to morphological examination. To view single cells in type I collagen matrix, the entire cell-containing 3D-col was stained with rhodamine phalloidin. As shown in Fig. 1B, HW and HV cells developed long protrusions extending from cell body, whereas HN cells possessed a compact morphology (Fig. 1B, panels a-c). Interestingly, HV cells displayed multiple membrane protrusions (Fig. 1B, panel c). Cell organization in 3D-col was visualized by phase-contrast microscopy (Fig. 1B, panels d-f). Consistent with the single cell morphology, HV cells were assembled into complex branching tubular networks (Fig. 1B, panel f). In contrast, HN cells failed to demonstrate organized structure (Fig. 1B, panel e).
Because the branching phenotype of carcinoma cells has been associated with their metastatic capacity (47), whether Rac1 mediates HT1080 cell invasion was assessed. Rac1V12 substantially increased, whereas Rac1V12N17 reduced, HT1080 cell invasion across 3D-col (Fig. 1C). To assess whether MMPs or plasminogen activator (PA)/plasmin system is responsible for this process, we monitored cell invasion in the presence of inhibitors for MMPs and PA/plasmin system, SC68180 and aprotinin, respectively. SC68180, formally known as SC44463, is a hydroxymate compound that has been shown to inhibit human keratinocyte migration on native type I collagen as a general MMP inhibitor (48). As shown in Fig.  1D, invasion of HW and HV cells through 3D-col was inhibited by SC68180. Interestingly, the active Rac1-induced cell invasion was reduced ϳ90% by the MMP inhibitor. In contrast, aprotinin did not impact on the collagen invasiveness. These data indicate that invasion of HT1080 cells across collagen depends on both Rac1 and MMP activities and that Rac1 requires MMP activities to promote cell invasion.
MMP-2 Is Differentially Activated in HT1080 Cells Stably Expressing Rac1 Mutants Cultured in 3D-col-To investigate the Rac1-mediated MMP activity in HT1080 cells surrounded by collagen matrix, we monitored MMP production by gelatin zymography in these cell lines when cultured in 3D-col for 24 h (Fig. 2A). The conditioned medium of HEp3, an epidermoid carcinoma cell line that secretes MMP-1, MMP-2, and MMP-9 as detected by gelatin zymography (49), was used as a control ( Fig. 2A, lane 1). Four major gelatinolytic bands with different intensity were detected in the serum-free medium of HW cell culture ( Fig. 2A, lane 2). These bands corresponded to the inactive MMP-9 proenzyme (92 kDa) and three MMP-2 species, latent (L), intermediate (i), and active (A). Interstitial collagenase MMP-1 was undetectable in HT1080-derived cell lines in gelatin ( Fig. 2A) as well as type I collagen zymography (Fig.  2B), a method that more sensitively detects MMP-1 than does MMP-2 (42). The distribution of three MMP-2 species was distinctly altered by the stable expression of Rac1V12N17 (HN) and Rac1V12 (HV). Active and latent MMP-2 became the major species in HV and HN cells, respectively ( Fig. 2A, lanes 2-4), suggesting Rac1 specifically mediates MMP-2 proenzyme processing cells cultured in 3D-col. Active MMP-9 was not detected in this system; instead, latent MMP-9 at 92 kDa was detected at low level in both HW and HV cells, but nearly undetectable in HN cells ( Fig. 2A).
Cells cultured in fibrin gel were used as control. Unlike 3D-col that induced MMP-2 processing, fibrin gel did not promote MMP-2 activation ( Fig. 2A, compare lanes 2 and 5). Predominantly latent MMP-2, as well as low level of the intermediate form, were detected in HW and HN cells in fibrin gel ( Fig.  2A, lanes 5 and 6). However, Rac1V12 induced MMP-2 activation by cells cultured in fibrin gel ( Fig. 2A, compare lanes 5 and  7). Similar MMP-2 activation pattern was also observed using type I collagen zymography (Fig. 2B). Taken together, these results suggest that Rac1 is required for maximal activation of MMP-2 by 3D-col and that active Rac1 is sufficient to induce MMP-2 processing in the absence of collagen signal.
To determine the specificity of Rac1 in mediating collageninduced MMP-2 activation, MMP-2 activating signals that are independent of Rac1 activity were sought. Concanavalin A (ConA) is known to activate MMP-2 (50). Stimulation of Rac1V12-or Rac1V12N17-transfected cells with ConA had similar effects on MMP-2 activation (Fig. 2C), suggesting that Rac1 is specifically involved in MMP-2 activation in response to type I collagen stimulation. Latent MMP-9 level remained similar in all three cell lines under this culture condition (Fig. 2C).
Rac1 Mediates Cell-associated Activation of MMP-2 by Type I Collagen-The activation of MMP-2 is thought to occur on cell membrane (51). To determine whether Rac1-dependent MMP-2 activation is associated with cells, cell-bound MMP-2 activity was assessed. Consistent with results from secreted MMP-2, the collagen-induced level of cell-associated active MMP-2 was greatly enhanced by Rac1V12, but reduced by Rac1V12N17 (Fig. 3A, lanes 4 -6). Active MMP-2 was also detected in cell extracts prepared from Rac1V12-expressing cells grown in fibrin gel, a condition that did not stimulate MMP-2 activation (Fig. 3A, lanes 1-3). Western blotting of cell extracts detected both proenzyme and processed MMP-2 in response to collagen or Rac1V12 (Fig. 3B), confirming the zymographic results (Fig.  3A). Therefore, we conclude that Rac1 mediates cell-associated activation of MMP-2.
Rac1 Mediates MT1-MMP Expression and Processing-Cellassociated activation of MMP-2 requires MT-MMPs (51). Among several MT-MMPs identified to data, MT1-MMP is the best documented in its ability to activate MMP-2. To assess whether Rac1 mediates MMP-2 activation through MT1-MMP, we first determined whether MMP-2 activation by Rac1V12 requires MMP activities. The synthetic MMP inhibitor SC68180 abrogated MMP-2 activation (Fig. 4, lanes 2 and 5), whereas the inhibition of serine proteinases/plasmin system by aprotinin did not (Fig. 4, lanes 3 and 6), suggesting that MMP activities were responsible for the cell membrane activation of MMP-2. The inhibitory effect of SC68180 on MMP-2 activation was observed in both cell and conditioned medium (Fig. 4).
Next, we assessed whether Rac1-mediated MMP-2 activation coincides with MT1-MMP expression. MT1-MMP mRNA was increased modestly by 3D-col (Fig. 5A, compare lanes 1 and  4). This induction was further enhanced by Rac1V12, but attenuated by Rac1V12N17 (Fig. 5, lanes 5 and 6). Rac1V12 had the ability to increase MT1-MMP mRNA in cells cultured in fibrin gel (Fig. 5A, lane 3). As shown in Fig. 5B, Western blotting detected two bands corresponding to 60 and 43 kDa, the two MT1-MMP species thought to represent the mature protein and its processing product, the N-terminal truncated protein, respectively (28,52). Two unspecified bands of high molecular weight were also detected by monoclonal antibody against MT1-MMP, presumably representing an artifact of the antibody. Although the level of the 60-kDa form was enhanced by Rac1V12, HW and HN cells demonstrated comparable level of the 60-kDa MT1-MMP (Fig. 5B). Interestingly, the level of 43-kDa protein, the truncated MT1-MMP that was suggested to reflect the consumption of MT1-MMP in the activation of MMP-2 proenzyme (28), mirrored the MMP-2 activation pattern as observed earlier (Fig. 2A). As shown in Fig. 5B, both  3, 4, and 6). Therefore, Rac1 mediated both MT1-MMP expression/processing and the accumulation of the 43-kDa MT1-MMP processing product in correlation with MMP-2 activation.
Tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), an effective inhibitor of MT1-MMP and membrane-associated MMP-2, is proposed to be a necessary component for MMP-2 activation at low concentration (23,27). The role of Rac1 in TIMP-2 expression was also determined. Results from Northern and Western blotting indicated that TIMP-2 mRNA and protein levels were not obviously changed by either collagen or Rac1V12 (data not shown). An increase in the amount of MT1-MMP and little change in the level of TIMP-2 protein by colla- gen or active Rac1 may imply a shift to increased cellular capability of activating MMP-2 proenzyme.
Rac1 Mediates Type I Collagen Fibril Degradation-Because both MMP-2 and MT1-MMP have the ability to degrade type I collagen (14,33,34), we wondered whether Rac1V12 might increase collagenolytic activity by HT1080 cells. To examine this possibility, fibrillar collagen degradation was assessed. Cells were seeded in a pellet on a reconstituted type I collagen fibril film and incubated in serum-free medium. Cells expressing vector control and Rac1V12 readily degraded the underlying collagen film to a different extent (Fig. 6A). Cells expressing Rac1V12N17, however, did not degrade as much the underlying collagen matrix. Interestingly, like 3D-col, the collagen fibril film also induced the processing of a fraction of MMP-2 proenzyme to active form in a Rac1-dependent manner (Fig.  6B). This process coincided with the modulation of collagenolytic activity.
To quantify the collagenolytic activity, cells were plated onto radioactive collagen fibril film. Released radioactivity indicated that collagenolytic activity in HW and HV cells was 1-5-fold higher than that in HN cells (Fig. 6C). Together these results demonstrated that Rac1 mediates collagenolytic activity in accordance with cell invasiveness (Fig. 1C), MMP-2 activation ( Fig. 2A and Fig. 3), and MT1-MMP expression/processing (Fig.  5, A and B).
Rac1-induced Cell Invasiveness in 3D-col Is Reduced by MMP-2 Inhibitors-The C-terminal hemopexin domain (CTD) of MMP-2 has been extensively used to specifically inhibit cell-associated MMP-2 activation (22,53,54) and to study MMP-2 activity-dependent cellular functions (20,36,55). In contrast, the collagen-binding domain (CBD) of MMP-2 that contains fibronectin type II-like modules does not have inhibitory effect on MMP-2 activation as stimulated by ConA (54,56). The recombinant CTD (Gly 417 -Cys 631 ) and CBD (Val 191 -Gln 364 ) were synthesized and tested for their ability to inhibit MMP-2 activation in HW and HV cells embedded in 3D-col (Fig. 7A). CTD inhibited MMP-2 activation in both cell lines in a concentration-dependent manner (Fig. 7A, lanes 1-6), whereas CBD did not have inhibitory effect (Fig. 7A, lanes 7 and 8). Therefore, CTD and CBD were used as a specific inhibitor of MMP-2 and a non-inhibitory control, respectively, in the collagen invasion assay.
To evaluate whether MMP-2 activity contributes to the invasive behavior of Rac1V12-expressing cells, we analyzed cell

FIG. 5. Stable expression of dominant negative and active Rac1 in HT1080 fibrosarcoma cells mediates MT1-MMP expression and processing in three-dimensional collagen matrix.
Three HT1080-derived cell lines were cultured in 3D-col or fibrin gels. Total RNA or cell extracts were prepared after 10 or 18 -24 h, respectively, for analysis by Northern (A) or Western (B) blotting. A, Northern blots were detected with cDNA probes for MT1-MMP or 36B4 mRNA, a ubiquitously expressed gene product (79). B, Western blots were detected with affinity purified monoclonal antibody against MT1-MMP. Two bands corresponding to molecular mass 60 or 43 kDa were indicated. Results are representative of at least three individual experiments.

FIG. 6. Stable expression of dominant negative and active Rac1 in HT1080 fibrosarcoma cells mediates collagenolytic activity.
A, three HT1080-derived cell lines were plated in the center of each well in a 24-well plate coated with a thin film of air-dried polymerized type I collagen. Cells were removed with typsin/EDTA after 3 days in serum-free medium and the residual collagen fibril film was stained with Coomassie Blue. Results are representative of three experiments. B, three HT1080-derived cell lines were plated in each well of a 24-well plate coated with a thin film of air-dried polymerized type I collagen. Serum-free medium was collected after 24 h and analyzed by gelatin zymography. C, three HT1080-derived cell lines were incubated in wells of a 48-well plate coated with 3 H-acetylated rat tail type I collagen. Samples of conditioned medium (50 l) were collected after 1 and 2 days, and the released radioactive degraded collagen was measured in a liquid scintillation counter. Net collagen degradation was quantified by subtracting the counts/min contained in 50 l of cell-free medium from each sample. The bar graphs represent the mean counts of triplicate samples in a single experiment. The similar experiment was performed four times with triplicate wells for each condition.
The inhibition of MMP-2 activation by various inhibitors coincided with the reduced cell invasiveness (Fig. 7B). As much as 80% inhibition of cell invasion was observed in the presence of TIMP-2. The presence of CTD resulted in ϳ60% reduction in cell invasion. However, TIMP-1 and CBD, two controls that did not inhibit MMP-2 activation, did not affect Rac1-induced cell invasiveness. Therefore, our results support the notion that modulation of MMP-2 activation is at least one of mechanisms by which Rac1 mediates cell invasion through type I collagen matrix barrier. DISCUSSION The Rho group of small GTPases and MMPs are two protein families that play key roles in cell movement. Here we report that Rac1-regulated cellular MMP-2 activation by type I collagen and that MMP-2 activity is required for Rac1-induced cell invasion across type I collagen barrier. In correlation with MMP-2 activation, Rac1 also modulated collagen-induced MT1-MMP expression/processing and collagenolytic activity. This suggests that Rac1 mediates the collagen-dependent MT1-MMP/MMP-2 activity as one of mechanisms by which it induces cell invasion.
Cells expressing dominant negative Rac1 reduced MMP-2 activation by 3D-col, implying that collagen fibril-cell interaction initiates a proteolytic cascade to cleave MMP-2 proenzyme through Rac1. To determine that MMP-2 proenzyme processing coincided with Rac1 activation in cells cultured in 3D-col, we performed the pulling down assay that selectively detects the GTP-bound Rac1. However, Rac1 activity was unchanged in vector-expressing cells stimulated by type I collagen (data not shown). It has been recently proposed that the GTP-bound small GTPases stimulated by selective ECM signals may be difficult to detect by the pulling down assay because the activated forms probably appear very transiently and are rapidly down-regulated (60). An alternative approach was thus taken to confirm the role of active Rac1 in MMP-2 activation by 3D-col. The stable expression of active Rac1 enhanced 3D-colinduced MMP-2 activation, confirming the results showing the suppression of MMP-2 activation by dominant negative Rac1. Furthermore, Rac1V12 conferred cells with the ability to induce MMP-2 activation and MT1-MMP expression/processing when cells expressing active Rac1 were cultured in fibrin gel, a matrix that does not induce MMP-2 activation. Therefore, we conclude that active Rac1 is necessary and sufficient for collagen-induced MMP-2 activation.
Our finding is in line with the study showing that MMP-2 activation is induced by 3D-col via ␤ 1 integrin (61). Cellular interaction with 3D-col has been shown to result in ␤ 1 integrin aggregation (62). This mode of ␤ 1 integrin modulation promotes MMP-2 activation/MT1-MMP processing (29) as well as phosphorylation of focal adhesion kinase (63). Activated focal adhesion kinase is linked to Rac1 by stimulating adaptor protein CAS (64), a molecule that promotes Rac1 activation after the complex formation with CrkII and DOCK180 (65)(66)(67). As expected from this chain of events (␤ 1 integrin aggregation 3 focal adhesion kinase 3 CAS/CrkII/DOCK180 3 Rac1), the role of Rac1 in MMP-2 activation may reflect that of ␤ 1 integrin aggregation. Indeed, we found that similar to ␤ 1 integrin aggregation, active Rac1 was sufficient for induction of MMP-2 activation. Furthermore, a blocking antibody against ␤ 1 integrin failed to inhibit Rac1V12-promoted MMP-2 activation in cells cultured in 3D-col, whereas ␤ 1 integrin aggregation-induced MMP-2 activation was attenuated by the stable expression of Rac1V12N17 (data not shown).
MMP-2 activation is unique among secreted MMPs in that it occurs on cell membrane (51). Membrane-bound MT1-MMP is known to initiate MMP-2 activation (24,26). It has been hy- FIG. 7. Rac1-promoted cell invasion through type I collagen requires MMP-2 activity. A, HT1080 cells expressing vector (HW) and Rac1V12 (HV) were cultured in 3D-col in the presence of CTD or CBD at the indicated concentrations. After 24 h, medium was collected and subjected to gelatin zymography using a 10% SDS-polyacrylamide gel electrophoresis. The results were representative of three individual experiments. B, Rac1V12-expressing HT1080 cells were added to the upper compartments of type I collagen invasion chamber in the presence or absence of the indicated components. After 24 h, an aliquot of the conditioned medium from the upper chamber was removed and analyzed by gelatin zymography. Cells that invaded to the underside of the filters were counted. Three invasion chambers were used per condition. Results shown are the mean of cell number in 10 randomly selected fields in a single representative experiment of six performed. FI, furin inhibitor; CTD, C-terminal domain; CBD, fibronectin type II-like modules.
pothesized that MT1-MMP and TIMP-2 form a "receptor" complex that binds MMP-2, resulting in the proteolysis of bound MMP-2 by an adjacent free MT1-MMP (23,27,68). This model predicts that the balance between TIMP-2 and MT1-MMP is of critical importance in determining the activation status of MMP-2. A variation in either TIMP-2 or MT1-MMP status could result in the modulation of MMP-2 proenzyme processing. Indeed, the culture of several cell types on or in 3D-col induces MT1-MMP expression and, correspondingly, an increase in MMP-2 activation (21,69). In line with these studies, our data showed that Rac1 induced cell-associated MMP-2 activation in correlation with altered level of MT1-MMP mRNA, but not TIMP-2 expression, implying that Rac1 activity facilitates a shift in balance toward increasing proteolytic activity of MMP-2. How might Rac1 mediate MT1-MMP expression that leads to MMP-2 activation? Active Rac1 has been previously reported to increase interstitial collagenase (MMP-1) expression in rabbit synovial fibroblasts through a series of events that include the induction of H 2 O 2 production, NF-B activation, and interleukin-1␣ secretion (70). Inflammatory cytokines including interleukin-1␣ and tumor necrosis factor-␣ can induce MT1-MMP expression in human endothelial cells (71). It is thus likely that in our cell system, Rac1 may regulate MT1-MMP expression and MMP-2 activation by modulating interleukin-1␣ production. However, the exact mechanism by which Rac1 mediates MT1-MMP expression and MMP-2 activation remains to be elucidated.
In addition to MT1-MMP expression, we also detected Rac1mediated MT1-MMP processing as judged by Western analysis. MT1-MMP proteins were detected as two major bands, 60 and 43 kDa. The accumulation of the 43-kDa MT1-MMP mirrored the status of MMP-2 activation mediated by active or negative Rac1. Recently, increasing evidence has indicated that the generation of a 43-kDa truncated MT1-MMP directly correlates with MMP-2 activation by signals such as phorbol 12-myristate 13-acetate (28,52,72), ConA (29), fibronectin (31), and 3D-col (30). In contrast, the level of 60-kDa MT1-MMP, most likely corresponding to the processed mature form of MT1-MMP (32), was not modulated by these stimulators. It is suggested that the 43-kDa form of MT1-MMP represents an inactive byproduct during the activation of MMP-2 proenzyme by active MT1-MMP, reflecting the consumption of MT1-MMP in the activation and release of MMP-2 (28). The spatial localization of MT1-MMP at invadopodia, a special membrane protrusion that is analogous to lamellipodia and makes contacts with the underlying ECM surface, is essential for its function in degrading ECM substrates (37). It is probable that MMP-2 activation by MT1-MMP also depends on the invadopodia localization (73). Therefore, the proper function of MT1-MMP may depend on both cellular expression and specific membrane localization. Rac1 may be involved in both processes. While it is conceptually unclear at this stage about the invadopodia structure in our three-dimensional tissue culture model, striking morphological differences among HW, HN, and HV cell lines were observed. We speculate that the Rac1-mediated spatial arrangement in 3D-col may be a functional switch for MT1-MMP in our cell system. This could explain, at least partially, that as reflected by the 43-kDa form level, only a fraction of cellular MT1-MMP is accessible to MMP-2. This possibility will be the subject of future investigation.
A number of experimental approaches have been taken to determine the function of MMP-2 in cell motility. These approaches include the specific inhibition with neutralizing antibody against MMP-2 and the C-terminal hemopexin domain of MMP-2 (36, 74, 75) and the indirect inhibition with furin inhibitor and antisense oligonucleotides to prevent processing and synthesis of MT1-MMP, respectively (36,59,76). We assessed the functional role of MMP-2 in Rac1-induced cell invasion by inhibiting MMP-2 activity with reagents that target at the membrane activation of MMP-2 (the C-terminal hemopexin domain of MMP-2), MT1-MMP processing (furin inhibitor), activity of both MMP-2 and MT1-MMP (TIMP-2), and activity of general MMPs (SC68180). The inhibition of MMP-2 activation correlated with the reduction of Rac1-mediated cell invasiveness, suggesting that MMP-2 activity contributes to this process. It should be pointed out that MT1-MMP and MMP-2 may mediate cell invasion as components of a proteolytic activity cascade as well as two individual type I collagen-degrading MMPs. This notion is consistent with our finding that suppression of both MT1-MMP and MMP-2 activity by furin inhibitor or TIMP-2 more effectively reduced cell invasion than the inhibition of MMP-2 activation alone by its C-domain. However, it is also possible that these two inhibitors might target at a broader range of molecules involved in cellular invasion than the specific MMP-2 inhibitor C-domain does. While the function of MMP-2 is studied in the context of its activator MT1-MMP, the direct contribution of MT1-MMP in Rac1-mediated cell invasion may be studied in a cell system that does not express MMP-2. By using MMP-2-negative cell lines, Koshikawa et al. (36,77) identified the MMP-2-independent function of MT1-MMP in cleaving laminin-5, a substrate of MMP-2. Whether native helical type I collagen can directly serve as a substrate of MMP-2 remains controversial (14,78). Whereas Aimes and Quigley (14) have shown the evidence that suggests MMP-2 as an interstitial collagenase, Seltzer and Eisen (78) have indicated that the helix-relaxed, but not native helical, type I collagen is susceptible to digestion by purified MMP-2. In our cell systems, it is yet to be defined whether MMP-2 is sufficient to degrade native type I collagen or requires the helix relaxation by another MMP to initiate type I collagen cleavage process.
In summary, we have shown that Rac1 mediated collageninduced MMP-2 activation and a shift in balance between MT1-MMP and TIMP-2 toward collagenolytic phenotype. Evidence was also presented that MMP activities, largely MT1-MMP and MMP-2, were necessary for Rac1-promoted cell invasion through 3D-col. These findings suggest a mechanism by which the encounter between type I collagen and invading tumor cells could stimulate elevated level of MMP-2/MT1-MMP activities, leading to increased collagenolysis. This reciprocal regulation between collagen and MT1-MMP/MMP-2 is at least partially responsible for the Rac1-mediated cell invasion across collagen barrier. Therefore, the block of an activity chain, type I collagen 3 Rac1 3 MT1-MMP 3 MMP-2, may impair the invasiveness of certain tumor cells in interstitial stroma.


s2fieldsofstudy: Biology Medicine

text:
Tumour-stromal interactions: Integrins and cell adhesions as modulators of mammary cell survival and transformation

Stromal–epithelial interactions modulate mammary epithelial cell (MEC) growth and apoptosis by influencing cell adhesion and tissue organization. Perturbations in the mammary stroma and cell adhesion characterize breast tumors and underlie the altered tissue organization, disrupted tissue homeostasis and enhanced survival phenotype of the disease. Apoptosis resistance likely arises during malignant transformation via genetic and epigenetic modification of cell adhesion pathways induced by a changing tissue microenvironment. Acquisition of adhesion-linked survival networks that enhance MEC viability in the absence of basement membrane interactions probably promote malignant transformation, and may render breast tumors sufficiently resistant to exogenous apoptotic stimuli to generate multidrug resistance.

Introduction
Homeostasis in the mammary gland is achieved by a balance between cell proliferation and cell death (apoptosis), which is reflected by an organized tissue structure. Breast cancer, which is a loss of tissue homeostasis, is characterized by perturbations in mammary tissue architecture, that is linked to alterations in the extracellular matrix (ECM) and in the adhesion molecules expressed by the MECs [1]. Correcting the adhesion defects in the mammary tumor epithelia can restore tissue organization and normal behavior to some breast tumor cells [2]. Conversely, altering the stromal microenvironment of the breast promotes the expression of tumorigenic potential in MECs [3]. This suggests that aspects of the breast cancer phenotype arise from alterations in the dynamic interplay between the epithelial cells, the mammary stroma, and the structural organization of the breast. Gaining an understanding of how disturbances in this relationship relate to the pathogenesis of human breast cancer will depend on delineating the subtleties of this dialog. This will require the application of appropriate model systems that can reconstitute stromal-epithelial interactions in the context of a three-dimensional tissue structure.
Breast cancers typically exhibit low rates of cell proliferation (apoptosis deregulation), often recur after years of dormancy (apoptosis evasion), and once re-established frequently acquire resistance to treatment (apoptosis Review Tumour-stromal interactions Integrins and cell adhesions as modulators of mammary cell survival and transformation resistance). As such, alterations in apoptosis probably predominate in the pathogenesis of human breast cancer. Therefore, an understanding of how the stroma influences adhesion and tissue architecture to modulate MEC survival, and how these pathways become deregulated in mammary tumors, should help to define the critical events that regulate breast cancer pathogenesis. In the present brief review, we discuss how altered stromal-epithelial interactions and changes in cell adhesion and tissue architecture influence MEC survival to drive malignant transformation in the breast. We then present data that suggest that the interplay between the tissue microenvironment, cell adhesion, and tissue architecture may also underlie the origins of the multidrug resistant breast tumor phenotype.

Cell adhesion and survival in mammary epithelial cells
Normal cells require adhesion to grow and survive, and anchorage independence for growth and survival is considered a key feature of transformed cells [4]. Consistent with this concept, primary and immortalized nontransformed human and murine MECs require adhesion to an ECM basement membrane (BM) in order to maintain their survival in culture. For example, both primary and immortalized MECs will retain their viability ex vivo in the presence of growth factors such as insulin-like growth factor-I and epidermal growth factor, provided that they are in contact with a laminin-rich BM [5]. This response is specific, because primary MECs on fibronectin or in a collagen I ECM will undergo apoptosis [6][7][8].
The mechanism by which adhesion to a laminin-rich BM mediates MEC survival is not completely known, but ligation and activation of the laminin receptor α 3 β 1 integrin is believed to be a part of the process [7,9]. Ligation of MEC β 1 integrins alters the activity of adhesion-associated kinases, such as focal adhesion kinase and integrin-linked kinase [4,6,10]. BM-mediated survival in MECs probably requires co-operative signaling with cytokine receptors, such as the insulin receptor [5] or the epidermal growth factor receptor (EGFR) [11]. Synergistic interactions between growth factor receptors and integrins in MECs presumably lead to the activation of downstream effectors such as phosphinositide 3-kinase (PI3-K), mitogen-activated protein kinase and/or nuclear factor-κB [6,12]. These enzymes in turn are functionally linked to pathways that can actively repress death by modulating the expression and/or activity of various apoptosis repressors, including members of the bcl-2 family [13]. In primary and immortalized murine MECs, for example, adhesion-dependent survival is associated with PI3-K induced repression of bax translocation to the mitochondria [14]. Moreover, integrin-linked kinase can stimulate Akt activity via PI3-K, and this in turn can influence murine MEC survival by altering the functional status of BAD [10,15].
Whether BM-directed integrin-linked pathways also operate to mediate MEC survival in the mammary gland has not been directly established. Nevertheless, there is good concordance between remodeling of the mammary gland in vivo and expression of genes associated with involution (apoptosis) [16]. Furthermore, loss of mammary gland function and apoptosis also correlate with increased expression of metalloproteinases, which are ECM-degrading enzymes. Indeed, parallel studies conducted ex vivo, using MECs ectopically expressing the metalloproteinase stromelysin-1 [17], directly demonstrated that acute exposure to metalloproteinases and rapid degradation of the BM would lead to apoptosis.
It should be noted that two waves of apoptosis occur during involution in the mammary gland. The first wave, or initiation stage, of apoptosis involves the death of a small population of differentiated MECs, and probably occurs as a consequence of changes in systemic hormones and/or mechanical forces. The second stage involves the death of the remaining acinar MECs, and is linked to the activation of metalloproteinases, ECM degradation, and the irreversible commitment to remodel the mammary gland [16]. The precise roles of cell adhesion and integrin signaling in either of these stages of involution have not been well defined. Nevertheless, it was reported [18] that chronic exposure of MECs to stromelysin-1 both in vivo and in culture resulted in malignant transformation, suggesting that MECs that circumvent BM-dependent survival are tumorigenic. Whether malignant transformation in the breast requires absolute independence from adhesion-linked survival cues is yet to be determined.

Anchorage independence for survival and malignant transformation of the breast
The perception that anchorage independence for survival is an essential feature of malignant breast tumors is consistent with reports that immortalized breast tumor cells are able to grow and survive in soft agar. More specifically, we and others have found that malignant human MECs no longer depend on ligation and activation of β 1 integrins for survival in culture [2, 7,9]. Using a tumor progression model called HMT-3522, in which it is possible to study the early changes that occur during malignant transformation [1], we found that as the nontransformed cells in this series progress towards malignancy, they gradually lose their dependency upon β 1 integrin for survival [7]. This suggests that circumvention of β 1 integrin adhesiondependent survival signaling may play a critical role in driving malignant transformation of the breast.
More recently, we determined that loss of β 1 integrin dependency for survival in this cell series is associated with a dramatic increase in the expression and activity of EGFR (Weaver et al, unpublished data). We also found that inhibiting the activity of the EGFR was sufficient to revert the malignant phenotype of the tumor cells and repress their anchorage independence for growth and survival [19]. Alterations in β 1 integrin-dependent survival and EGFR activity occur concomitantly with a perturbed ability of the HMT-3522 cells to form breast tissue-like structures in response to a reconstituted BM (Weaver et al, unpublished data). This emphasizes the existence of an association between cell adhesion-directed tissue architecture, growth control, and apoptosis regulation in MECs. These findings also imply that deregulation of this relationship could lead to malignant transformation.

Is anchorage independence necessary for malignant transformation in mammary epithelial cells?
Clinical data support the idea that independence from BM-directed survival is linked to malignant transformation in the breast. Immunologic studies [20] have shown that invasive breast tumor cells exhibit a reduced level of apoptosis when compared with cells located in benign ductal carcinoma in situ (DCIS) lesions. Similarly, intense staining for focal adhesion kinase, a tyrosine kinase that can induce anchorage-independent survival in epithelial cells, was detected both in invasive tumor cells and in groups of premalignant cells within adjacent DCIS lesions [21]. Unfortunately, these data do not establish whether the enhanced survival in the transformed cells is due to genetic selection or is mediated via microenvironmental factors.
Although genetics undoubtedly plays a critical role in driving malignant transformation and apoptosis resistance in the breast, evidence is slowly accumulating that microenvironmental factors must also play a role in these processes. For example, angiogenesis can enhance mammary tumor viability, irrespective of genetic selection [22], whereas nonmalignant MECs exposed to a reactive stromal ECM can be induced to develop a tumor-like behavior in the absence of genetic events [3]. Indeed, stromal fibroblasts associated with mammary tumors have been shown to display a 'fetallike' behavior, and this altered phenotype has been suggested to modify significantly the kinetics of tumor progression [23]. Interestingly, data show that primary human breast tumors frequently exhibit a decrease in the expression of the 'differentiation-associated' laminin/collagen integrin receptors α 2 , α 3 , and β 1 , but they often express the 'invasion and growth-linked' tenascin and fibronectin receptors α v integrin and α 5 integrin [1]. Some aggressive breast tumors even retain expression of the laminin integrins α 6 and β 4 , and secrete BM proteins [24].
Because MECs within DCIS lesions seldom show changes in their integrin expression, this indicates that the dramatic shifts in integrin expression may be necessary to support tumor cell survival and drive malignant transformation. Although one could argue that the changes in integrin expression are solely due to selection of a genetically variant population of cells, it is also possible that the altered integrin expression observed in breast tumors reflects a dynamic adaptive survival response by the tumor cells to the interstitial stromal ECM. This would depend on the ability of the tissue microenvironment to modulate integrin expression and apoptosis resistance epigenetically in MECs.
In support of this concept, significant and rapid changes in integrin expression have been documented in primary tumor cells before (in tumors in situ that are in contact with a reactive stromal ECM) and after growth within a reconstituted BM ex vivo [25]. We and others [19,26,27] have also observed that the ECM microenvironment can dynamically modulate integrin expression in both primary and immortalized MECs. Most recently, we found that malignant transformation and β 1 integrin independence in the HMT-3522 tumors occurs in conjunction with, and is dependent on, ligation of α 6 β 4 integrin and secretion of BM protein (Zahir et al, unpublished data). These results are consistent with reports that high levels of expression of α 6 and β 4 integrins, and BM proteins in human breast carcinomas correlate with reduced patient survival, and functional deletion of α 6 integrin in metastatic breast tumor cells results in a significantly higher rate of apoptosis [24,28,29]. Therefore, a more realistic interpretation regarding the evolution of apoptosis resistance and malignant transformation in the breast is that it arises by a combination of genetic 'mutation/selection' pressures and epigenetic 'adaptation' responses induced by the tissue microenvironment.
Studies conducted with isolated primary human breast tumor cells support the idea that stromal-epithelial interactions are primarily responsible for promoting survival in primary breast tumors in vivo, as opposed to cell autonomous 'selection' events. 'Tumorigenic' breast cell lines isolated from primary breast tumors that represent earlier, less aggressive breast cancer phenotypes do not always exhibit true anchorage independence for growth and survival. Indeed, the majority of immortalized tumor cells used to study apoptosis regulation and anchorage independence in human breast cancer have been generated from late-stage disease cells isolated from metastatic pleural effusions, in which stromal interactions are minimal and cell-cell interactions are predominant [30]. Along this vein, Giovanella et al [31] reported that a mere 6.1% (16/262) of primary infiltrating duct-cell human breast carcinomas survived and grew following injection into nude mice. Only cells from those tumors that were highly cellular (enhanced cell-cell interactions) and lacked detectable desmoplastic hyperplasia (minimal stromal involvement) could be grown and serially transplanted into nude mice.
Interestingly, primary breast tumor cells that are first embedded within purified extracellular collagen I or a reconstituted BM can be successfully grown and propagated in nude mice [32]. It has also been observed that primary human breast tumor cells can be maintained and effectively studied ex vivo if the cells are maintained in the presence of either a reconstituted BM or purified collagen I matrix [33,34]. Therefore, a more prudent conclusion regarding malignant transformation and apoptosis resistance in breast cancer is that, rather than acquiring absolute anchorage independence for survival, most primary human mammary tumors probably depend on altered stromal factors and/or adhesive interactions to maintain their viability in vivo.

Cell adhesion, tissue architecture and apoptosis resistance in breast cancer
Breast tumors characteristically lack tight junctions [35], and exhibit disrupted E-cadherin organization [1]. These observations are consistent with the idea that loss of cell-cell adhesion is essential for tumor invasion [36]. However, aggressive breast tumors with a poor prognosis often upregulate different cell adhesion molecules such as P-cadherin, CD44, and Ep-Cam [37][38][39], and readily aggregate. Enhanced cell-cell interactions can repress apoptosis [40], and the success of culturing primary breast tumor cells ex vivo in three dimensions may be explained in part by the augmentation of nonclassical intercellular communication in the spheroid cultures [41]. A relationship between multicellular-mediated drug resistance, the metastatic phenotype, and cell adhesion has also been established [42]. These findings suggest that altered intercellular communication in breast tumors may be another mechanism whereby BM independence and apoptosis resistance can be generated in breast tumors.
It is not known how intercellular interactions support cell survival. However, Carmeliet et al [43] showed that VEcadherin functionally interacts with the vascular endothelial growth factor receptor, PI3-K, and active Akt in a physical complex to mediate endothelial cell survival in vivo. Green fluorescent protein tagged protein kinase B/ Akt, which facilitates adhesion directed survival in MECs, also localizes to both cell-ECM and cell-cell junctions [44]. Furthermore, the retinoblastoma protein, which is implicated in ECM-directed survival, mediates adherensjunction-dependent survival in MECs [45]. Thus cell-cell adhesion probably supports MEC survival by actively repressing apoptosis via cross-talk with growth factor and ECM-linked survival pathways.
We and others have shown that long-term survival and apoptosis resistance in MECs in a reconstituted BM is dependent on the formation of a multicellular tissue-like structure and the assembly of adherens junctions (Weaver et al, unpublished data) [46]. Studies have shown that acquisition of the multidrug-resistant phenotype can be significantly accelerated if the tumor cells being studied are grown as three dimensional spheroids [42]. Tumor cells grown as three dimensional spheroids exhibit enhanced cell-cell communication, change their integrin expression, and secrete ECM proteins [41,47]. This indicates not only that cell-cell interactions could modulate ECM-directed survival via integrin and growth factor receptor mediated events, but also that such cross-talk may additionally confer apoptosis resistance on MECs.

Conclusions
The molecular basis for the cross-modulation of survival networks by cell-cell and cell-ECM interactions remains ill defined. Cells dramatically reorganize their cytoarchitecture when cultured as three dimensional apoptosis resistant spheroids, however, and perturbing cytoskeletal organization restores their apoptosis sensitivity (Weaver et al, unpublished data). Because actin modifiers can regulate cell survival [48], and are themselves altered during apoptosis [49], this indicates that the regulation of apoptosis by cell adhesion cross-talk probably depends on the cytoskeleton and its molecular regulators. Nevertheless, the existence of dynamic and reciprocal cross-modulation of apoptosis by cell-cell and cell-ECM implies that any significant change in the breast stromal ECM, or in the adhesion molecules expressed by the breast epithelium, will have some impact on MEC survival and/or apoptosis sensitivity. Should alterations in any one or more of the adhesion-linked apoptosis regulatory network components confer enough of a survival advantage to the cells, then this could favor MEC viability in the absence of a BM, and thereby promote malignant transformation. If the changes were of sufficient magnitude, then they could even render some breast 'tumor' cells resistant to exogenous apoptotic stimuli, and hence give rise to the multidrug-resistant breast cancer phenotype. Indeed, this paradigm can explain the profound effects exerted by a reactive stroma on cancer progression, and why the site of tumor metastasis can exert such a strong influence on the efficacy of cancer treatment.