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Cell Growth & Differentiation Vol. 12, 147-155, March 2001
© 2001 American Association for Cancer Research

Apoptotic Signaling during Initiation of Detachment-induced Apoptosis ("Anoikis") of Primary Human Intestinal Epithelial Cells1

Johannes Grossmann2, Kathrin Walther, Monika Artinger, Stephan Kiessling and Jürgen Schölmerich

Department of Medicine I, University of Regensburg, 93042 Regensburg, Germany


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Apoptosis after the loss of cell anchorage—"anoikis"—plays an important role in the life cycle of adherent cells. Furthermore, loss of anchorage dependency is believed to be a critical step in metastatic transformation. The aim of this study was to further characterize the sequence of intracellular events during anoikis in a nontransformed population of human intestinal epithelial cells (IECs). Purified human IECs were kept in suspension to induce anoikis in over 90% of IECs within 3 h. Two initiator caspases, caspase-2 and -9, are activated within 15 min, followed by the hierarchical activation of downstream caspases within 1 h. The activation of the caspase FLICE (caspase-8) does not contribute to the initiation of anoikis, and massive release of cytochrome c from mitochondria cannot be detected before 60 min, indicating that cytochrome c release does not play a role during initiation of anoikis. This study delineates the signaling cascade during anoikis of nontransformed cells. Future studies may identify alterations of this cascade in neoplastic cells, thereby possibly gaining insight into carcinogenesis and metastatic transformation.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Apoptosis, the programmed execution of cell death, is a fundamentally important process. Beyond regulating tissue homeostasis, "too much" or "too little" apoptosis is responsible for a rapidly growing list of diseases. Tight regulation of cell death to maintain homeostasis is of particular importance for cells with short life cycles and high rates of proliferation, such as IECs.3

IECs are generated by stem cells at the crypt base and migrate toward the intestinal lumen within only 3–5 days (1) . At the villus tip (in the small bowel) or luminal surface (in the large bowel), respectively, their short life cycle is terminated as these cells undergo apoptosis, loose anchorage, and shed into the lumen (2, 3, 4) . Induction of apoptosis by loss of cell anchorage, a form of apoptosis termed "anoikis" (5) , is likely to be one the mechanisms terminating the physiological life cycle of these cells because IECs seem to gradually loosen cell anchorage approaching the lumen: changes of integrin expression and cadherin binding, as well as the changing expression of basement membrane components, mediate an antiadhesive environment, culminating in detachment of the dying enterocyte at the villus tip/luminal surface (6, 7, 8, 9, 10, 11) . However, the molecular mechanisms of IEC apoptosis/shedding and the means of cell death execution poorly characterized. This process needs to be highly regulated and fast to maintain integrity of the intestinal epithelium.

Caspases, a newly defined group of cysteine proteases (12) , play a central role during apoptosis. They mediate the intracellular activation of other caspases and selectively cleave distinct intracellular substrates, leading to dismantling of a cell’s architecture, signaling apparatus, and repair mechanisms. Eventually, caspases induce the activation of endonucleases that complete cellular suicide by internucleosomal DNA fragmentation (13, 14, 15) . Caspases are divided into two groups, based on their assumed role in the apoptotic cascade (reviewed in Ref. 16 ). Whereas long-prodomain caspases (caspase-2, -8, -9, and -10) are regarded as "initiator caspases" functioning upstream in the cascade, short-prodomain caspases (caspase-3, -6, and -7) are regarded as downstream "executioner caspases." Similar to the complement system, caspases are believed to become activated in a hierarchical cascade, yet the exact mechanisms of activation and the sequence of events are still incompletely understood. Studies on the cascade of caspase activation during receptor-mediated apoptosis, radiation-induced apoptosis, or chemically induced apoptosis show that distinct cascades might be activated, depending on the form of apoptosis being executed (17, 18, 19, 20) . During certain forms of apoptosis and in certain cell types, the mitochondrial release of cytochrome c leading to the formation of the so-called apoptosome seems to play an important role as an initiating and augmenting factor of apoptotic signaling (21, 22, 23) .

Programmed cell death can be induced by several means, one of which is loss of cell anchorage in adherent cells. Meredith et al. (24) first described endothelial cells dying of apoptosis due to loss of anchorage in 1993, and Frisch and Francis (5) proposed the term anoikis for this form of apoptosis. Anoikis has been described for an array of adherent cells including mammary cells (25 , 26) , kidney cells (5) , thyroid cells (27) , intestinal cells (28 , 29) , bronchial epithelial cells, keratinocytes (30 , 31) , endothelial cells (32) , and fibroblasts (33) . Apart from its relevance in physiological circumstances such as mammary gland involution, postinflammatory capillary involution, and IEC physiology, loss of anchorage dependency is believed to be one of the key mechanisms leading to metastatic transformation of a neoplastic cell because it will enable a malignant cell to detach and spread from the primary tumor without undergoing anoikis (5 , 34, 35, 36, 37) .

Anoikis can be induced by loss of cell-matrix contact and/or loss of cell-cell contact (5 , 28 , 33 , 38, 39, 40, 41, 42) ; however, the molecular mechanisms initiating anoikis are still incompletely understood. FAK, an integrin-associated non-receptor kinase, seems to play a pivotal role transducing cell-matrix-mediated survival signals (43) . Loss of FAK signaling rather than caspase-mediated cleavage of FAK seems to induce activation of the apoptotic signaling cascade (38 , 43 , 44) . Caspase-9 can be activated due to cessation of protein kinase B/Akt-mediated phosphorylation (45) ; Akt is a downstream element of the phosphatidylinositol 3'-kinase (PI-3K) pathway, which plays a role in integrin/FAK-mediated survival signaling (46) . E-cadherins mediate cell-cell anchorage in IECs by homotypic zipper-like binding. Mutations or down-regulation of E-cadherin leads to anchorage independence and malignant transformation (47) , but the link between physiological E-cadherin signaling and inhibition of apoptosis is largely unknown. E-cadherin binding will lead to increased phosphorylation of proteins at sites of cell-cell contact (48) . However, phosphorylation of the E-cadherin-associated ß-catenin will promote its proteasome-mediated degradation, thereby blocking its interaction with transcriptional activators lymphocyte-enhancing factor 1 and T-cell factor 4 targeting genes such as c-myc (49) . The mechanisms leading to activation of the apoptotic cascade due to loss of cell-cell contact are unknown.

Some studies attribute a pivotal role to stress-activated protein kinases during anoikis signaling (50 , 51) , a finding that has not been confirmed by other investigators (52 , 53) . Recently, two groups concluded that anoikis is initiated via Fas-associated caspase-8 (54 , 55) , based on the observation that transfection of epithelial cell lines with dominant negative Fas-associated protein with death domain (FADD) was able to block anoikis.

In extension of our previous work on anoikis (29 , 56) , a comprehensive study on apoptotic signaling events during anoikis, such as long-prodomain caspase activation, mitochondrial cytochrome c release, intracellular translocation of caspases, and induction of DNA fragmentation, is presented, thereby describing additional characteristics of the anoikis pathway. In nontransformed primary human IECs, anoikis involves a highly ordered sequence of apoptotic signaling events initiated by the activation of procaspase-2 and -9 and followed by cytochrome c release and activation of caspase-8, which is clearly not the initial caspase to be activated during IEC anoikis.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Primary Colonic IECs Are Highly Susceptible to Anoikis Independently of Their Location on the Crypt-Lumen Axis.
Isolated cells were first stained with EP-4, anti-CD-3, and anti-CD-33 (cell surface markers of epithelial cells, lymphocytes, and macrophages, respectively) to ensure IEC purity by flow cytometry. To perform single cell analysis, isolated intact crypts were treated briefly with dispase before fixation.

A total of 97 ± 2% of primary cells harvested stained positive for the epithelial cell marker EP-4 (Fig. 1A)Citation , whereas hardly any cells stained positive for CD3 (1 ± 0.5%, Fig. 1BCitation ) or CD33 (1 ± 0.5%, Fig. 1CCitation ), demonstrating that our protocol ensured isolation of highly purified IECs. Isotope control staining was negative (Fig. 1D)Citation .



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Fig. 1. Isolation of highly purified primary human IECs. IECs were isolated as described, and purity was assessed by flow cytometry. Nearly 100% of cells showed positive staining for the epithelial cell marker EP-4 (A), whereas less than 2% of cells stained positive for CD-3 (B) or CD-33 (C). Isotype control staining was negative (D). Data are representative of five experiments.

 
In previous studies we showed massive DNA laddering by DNA gel electrophoresis after 90 min of induction of anoikis (29) . To further quantify these data beyond the microscopically scored ethidium bromide/acridine orange stain (29) , IECs were harvested at different time points after loss of anchorage and analyzed for evidence of DNA fragmentation by PI stain and flow cytometry. After pulse width gating to exclude the remaining cell aggregates, we could confirm that IECs rapidly undergo apoptosis within 3 h of loss of anchorage as depicted by a massive sub-G1 peak (92 ± 3% at 180 min) after loss of anchorage (Fig. 2)Citation .



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Fig. 2. After loss of cell anchorage, IECs rapidly undergo massive apoptosis. IECs were harvested and assessed for DNA content by PI staining. The cell aggregates remaining after a brief dispase treatment were identified and gated in the pulse width count. At the indicated times after loss of anchorage, PI staining of IECs was analyzed, showing a massive increase in the sub-G1 (M1) population of cells after 180 min undergoing anoikis, indicative of apoptosis.

 
To assess whether IECs of different regions along the crypt-lumen axis are differentially susceptible to anoikis, we isolated IECs from each of these regions using an established method described by Mayer et al. (57) . IECs underwent anoikis after loss of cell anchorage independently of their location along the crypt-lumen axis, as shown by DNA electrophoresis of IECs after 3 h in suspension (Fig. 3A)Citation . To quantify this observation, we measured caspase-3-like activity at identical times. IECs of different regions along the crypt-villus axis showed no difference in caspase-3-like activity (Fig. 3B)Citation . These findings demonstrate that freshly detached primary human IECs are equipped to execute massive programmed cell death within 3 h. Nontransformed human IECs are genuinely susceptible to the induction of anoikis independently of their state of differentiation or proliferation.



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Fig. 3. IECs are susceptible to anoikis independent of their state of differentiation. IECs were isolated from different regions along the crypt-lumen axis (CB, crypt base; MI, mid-crypt; LU, lumen region of crypt) and kept in suspension for 3 h to assess susceptibility to anoikis. As shown by DNA electrophoresis (A) and caspase-3-like activity (B), no difference can be detected between IECs of different developmental stages. Data are mean ± SD of three experiments. M, 100-bp ladder.

 
Rapid Activation of Caspase-2 and -9, but not Caspase-8, during Initiation of Anoikis in Primary Human IECs.
Caspases with long prodomains function as initiator caspases upstream in apoptotic cascades. Depending on the trigger of apoptosis, the caspase(s) mediating the initiation of the caspase cascade may vary. Because the initiator caspases of anoikis are unknown, we performed Western blots to specifically assess the activation of long-prodomain caspase-2, -8, -9, and -10. In contrast to our previous studies showing that caspase-10 remains inactive during anoikis (29) , we show in Fig. 4Citation that caspase-2 is activated within 15 min of isolation (initiation of anoikis) because the prodomain is rapidly cleaved off, and the p16 subunit of caspase-2 becomes detectable. Caspase-9 also undergoes rapid processing within 15 min of detachment as the prodomain is lost, yielding the p37 intermediate that is rapidly further processed to yield the p10 subunit depicting the fully activated caspase within 30 min of detachment. Interestingly, caspase-8 remains initially inactivated (Fig. 5)Citation ; however, after 30 min, the prodomain of caspase-8 is processed, and at 45–60 min after loss of cell anchorage, the p16 subunit indicative of activated caspase-8 is detectable.



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Fig. 4. Caspase-2 and caspase-9 function as upstream caspases during anoikis. IEC cytosol was extracted at the indicated times during anoikis and analyzed by Western blot for activation of caspases. Caspase-2 (p48) is activated almost instantly by deletion of the prodomain (yielding the p33 intermediate) as the active p16 becomes detectable before 15 min of detachment. Caspase-9 is processed within 15 min as the prodomain is lost, yielding the p37 intermediate, and further rapid processed to yield the p10 subunit within 15–30 min. The protein at Mr 35,000 represents another intermediate of caspase-9 activation.

 


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Fig. 5. Caspase-8 shows delayed activation during anoikis. IECs were processed as described above. Caspase-8 (p48 and p45 isoforms) is preformed in IECs and remains initially inactivated during anoikis. Thirty min after loss of anchorage, the prodomain of caspase-8 is processed (yielding the p43 and p41 fragments), and the p16 subunit of activated caspase-8 is detectable after 45–60 min.

 
These data show that anoikis initiation involves the activation of two long-prodomain caspases, caspase-2 and caspase-9, within the first 15 min of loss of anchorage. Initiator caspases are activated selectively and hierarchically, with caspase-8 showing a delayed activation, demonstrating that the initiation of the anoikis cascade is not mediated through caspase-8.

Anoikis Involves the Release of Cytochrome c from Mitochondria.
Release of cytochrome c in conjunction with loss of mitochondrial membrane potential has been described for distinct cell types and certain forms as the initiating event of apoptosis (17 , 18) . However, the role of mitochondrial cytochrome c release, which is also thought to serve as an intracellular augmentation factor of apoptosis (23) , in anoikis is unknown. Therefore, primary human IECs were lysed without sonication using a weak detergent facilitating the separation of distinct intracellular compartments by centrifugation. As shown in Fig. 6ACitation , cytochrome c is indeed released into the cytosol as primary human IECs undergo anoikis. Whereas minimal cytochrome c is released before 30 min of the cascade, there is a massive cytochrome c release after 60 min of anoikis. Equal loading of cytosolic samples was demonstrated by reprobing the identical gel for actin. Negative staining of cytosol for cytochrome c oxidase subunit IV demonstrated a lack of contamination by mitochondria. Mitochondria showed staining for cytochrome c at any time during the apoptotic cascade (data not shown), and staining for cytochrome c oxidase subunit IV was positive, assuring that purification of mitochondria was successful (Fig. 6B)Citation . These data demonstrate that the execution of IEC anoikis involves the release of cytochrome c from mitochondria. Because only trace amounts of cytochrome c are released during the initiation phase, followed by massive release during the execution phase, it is unlikely that cytochrome c release plays a major role during the initiation of anoikis, but it is rather an important element of the downstream cascade, augmenting apoptotic signaling.



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Fig. 6. Cytochrome c release is not the initiating event during IEC anoikis. IECs were processed at the indicated time points, and cytosolic and mitochondrial fractions were separated. A, IEC cytosol shows no cytochrome c (cyt. c) after isolation, and minimal amounts can be detected in the cytosol during the initial 30 min of anoikis (Western blot). However, cytochrome c is massively released after 60 min. Equal loading of cytosolic samples was demonstrated by probing the identical gel for actin. B, no detection of cytochrome c oxidase subunit IV demonstrated a lack of contamination by mitochondria in the cytosol by Western blot (c). Staining for cytochrome c oxidase subunit IV was positive in mitochondrial extracts (m), assuring us that purification of mitochondria was successful.

 
Intracellular Translocation of Activated Caspase-3 during IEC Anoikis.
Caspase-3, a pivotal downstream caspase, is activated after 30–45 min of anoikis (29) . Using a novel antibody that detects only activated caspase-3 (CM-1), we extended our studies on caspase-3 activation during the cascade of anoikis by performing antiactivated caspase-3 immunocytochemistry on IECs. Whereas only a minimal fraction of freshly isolated IECs display evidence of caspase-3 activation (data not shown), within 45 min of anoikis, activated caspase-3 is detected in IECs displaying a diffuse cytosolic staining.

After 90 min, activated caspase-3 can be detected predominantly in nuclei or evolving nuclear fragments (Fig 7)Citation . These findings confirm that caspase-3 is also activated during IEC anoikis and undergoes translocation as the apoptotic cascade proceeds, shifting from a predominant cytosolic localization to the nucleus of the dying enterocyte.



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Fig. 7. Translocation of activated caspase-3 during anoikis. IECs were harvested after 45 and 90 min during anoikis and analyzed for detection of activated caspase-3 by immunocytochemistry. Note the shift of brown staining for activated caspase-3 from a predominant cytosolic location (+45 min) to a the strong demarcation of apoptotic nuclear beads during late stages of apoptosis (+90 min).

 
Hierarchical Activation of Downstream Caspase-7, -3, and -6 and Cleavage of DFF during IEC Anoikis.
In our previous studies, we showed the activation of downstream caspase-7 and -3 after 15–30 and 30–45 min, respectively (29 , 56) . Furthermore, here we performed Western blot analysis for caspase-6 using a novel and improved antibody recognizing the procaspase as well as the p17 subunit. Fig. 8Citation demonstrates that caspase-6 activation occurs late during the apoptotic cascade as the p17 subunit becomes detectable after 60–90 min of anoikis.



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Fig. 8. Activation of short-prodomain caspase-6. IEC cytosol was processed as described above and analyzed for activation of short-prodomain caspase-6 by Western blot. Note the appearance of the subunit of caspase-6 (p17) after 60 min as the procaspase signal (p34) diminishes, showing that caspase-6 activation during anoikis is a late event during the apoptotic cascade.

 
Caspase-3-mediated cleavage of DFF links the caspase cascade to DNA fragmentation (58) . Because the fate of DFF during anoikis is unknown, cleavage of this caspase substrate was also assessed. In accordance with the data on DNA fragmentation and caspase activation shown above, cleavage of DFF could be detected 30–45 min after initiation of the apoptotic cascade (Fig. 9)Citation .



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Fig. 9. Cleavage of DFF during anoikis. Initiation of DNA fragmentation was verified on the protein level by assessment of DFF cleavage, a known caspase substrate by Western blot. DFF (p45) is cleaved to yield the caspase signature fragment (p30) after 30 min, before the initiation of DNA fragmentation by endonucleases.

 
These findings show that downstream caspases are activated in a hierarchical manner during anoikis. Primary IEC anoikis is executed by the sequential activation of the downstream caspase-7, -3, and -6. Completion of the apoptotic cascade by DNA fragmentation during IEC anoikis is mediated by cleavage of DFF, which is concurrent with the activation of the downstream caspase-3, which is known to mediate DFF cleavage in other forms of apoptosis.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Anoikis is a form of apoptosis described for a wide array of cells that need survival signals through cell anchorage to survive. The cessation of these anchorage-mediated signals seems to be a common mechanism to physiologically terminate the life cycle of these cells by apoptosis. Beyond its relevance for tissue homeostasis, anoikis is assumed to be of critical importance for metastatic transformation. The acquisition of anchorage independence, i.e., resistance to anoikis, is assumed to be a pivotal step during malignant transformation allowing tumor cells to become metastatic because anchorage-dependent normal or nonmetastatic tumor cells will die if they detach from the primary tumor to enter blood or lymphoid circulation.

Given its physiological as well as pathophysiological relevance, research on the molecular mechanisms mediating anoikis will enhance our understanding of epithelial cell physiology and may depict new avenues to fight neoplasia.

The focus of this study was to delineate the intracellular cascade of initiating apoptotic signaling events during anoikis with special reference to the caspase cascade. We have developed a novel model, isolating highly purified nontransformed human IECs to pursue these studies in so-called "primary" cells. What conclusions can be drawn from this study, and how do they enhance our understanding of IEC biology, anoikis, and apoptosis?

The time course of anoikis in these primary IECs is striking, and, to our knowledge, few cells will execute apoptosis with similar kinetics. IECs are obviously equipped to instantly execute anoikis. On loss of anchorage, IECs will immediately activate caspases and execute apoptosis in essentially all IECs to the level of DNA fragmentation within 3 h. These data may reflect a characteristic of primary IECs because anoikis of transformed cell lines or cells with a slower physiological turnover takes several hours (5 , 41) or days (25 , 32 , 33) before detachment-induced programmed cell death is executed. All caspases tested are preformed within IECs and abundantly expressed, and their activation during anoikis does not require de novo protein synthesis (5 , 29) .

Given the stronger expression of antiapoptotic bcl-2 family members in IECs at the base of the intestinal crypt, our finding that these cells are just as sensitive to anoikis as those close to the region of shedding seems somewhat surprising; however, it may reflect that loss of cell anchorage is an inevitable death signal, incompatible with life for any nontransformed IECs.

The data presented give new insight into the molecular events during the initiation phase of the anoikis signaling cascade. Our findings cannot confirm the assumption drawn by other investigators that long-prodomain caspases do not play a role during anoikis (59) . Indeed, two long-prodomain caspases, caspase-2 and 9, are activated within 15 min of IEC anoikis, before the massive release of cytochrome c from the IEC mitochondria. These data demonstrate that anoikis follows a cascade similar to that of death receptor-mediated apoptosis rather than a pattern seen in chemically and radiation-induced apoptosis, where mitochondrial events occur upstream of caspase activation (17 , 18) . Seemingly in agreement with this assumption is the finding of two recent studies suggesting that caspase-8 may be the initial caspase activated via Fas-associated death domain during anoikis (54 , 55) . Indeed, caspase-8 is activated during IEC anoikis, but we could demonstrate by Western blot that caspase-8 is not the first caspase to be activated during anoikis in primary IECs. In contrast to the other reports, the antibody used in this study recognizes procaspase-8 as well as the p18 subunit indicative of the mature caspase. Our Western blots show that caspase-8 remains inactive during the initial 30 min, and the p18 subunit becomes detectable when 45–60 min have passed. Two explanations may account for these differences. First, IECs are isolated in our system within an intact crypt structure, and it takes about 15–30 min until the crypts and consequently the cell-cell contacts are fully disrupted. Therefore, as speculated by Frisch (54) , significant changes of IEC shape, possibly leading to "induced proximity" of death receptors and consecutive activation of caspase-8 (60) , may be delayed until cell-cell contact is lost. Second, the cascade of anoikis may vary between cell types or may reflect differences of intracellular signaling between immortalized cell lines and primary epithelial cells. During anoikis of IECs, caspase-8 activation is a downstream event, and our data suggest that it may reflect a positive feedback mechanism serving as an additional amplification loop.

Short-prodomain caspase-7, -3, and -6 are hierarchically activated during anoikis, with caspase-7 upstream of caspase-3 and caspase-6 activation. Indeed, caspase-6 is activated at the end of the cascade, a finding that does not confirm our previous observation (29) but demonstrates that activation of a specific caspase should not be assessed by any fluorogenic substrates but only by Western blot using antibodies that detect the proform and the subunits generated during activation. Cytochrome c is released from mitochondria during anoikis, presumably as an augmenting factor during the execution phase, when massive amounts are released, whereas the trace amounts of cytochrome c that can be detected eary during anoikis are unlikely to initiate anoikis. Indeed, the previously shown surge in caspase activity (29) is cotemporal with the massive release of cytochrome c. Finally, this study contributes to the understanding of caspase trafficking within a cell during apoptosis. In agreement with the observations of Chandler et al. (61) , caspase-3 initially shows a strong cytosolic activation pattern but displays a shift to the nucleus and its remnants during later stages of the apoptotic cascade.

In summary, this work further describes the sequence of intracellular events during anoikis, a physiological form of apoptosis, in a population of primary epithelial cells. Whereas our studies follow the caspase cascade upstream, the initiating event leading to the activation of caspase-2 is still unknown and is being addressed in ongoing studies. However, the data presented delineate the presumptive "default" cascade of apoptotic signaling during anoikis of primary cells (Fig. 10)Citation . To our knowledge, this work contributes to the few studies describing an apoptotic cascade in nontransformed human cells. Future studies on malignant and premalignant primary IECs are underway that might delineate the steps of the anoikis cascade at which alterations have occurred, rendering these cells anchorage independent and possibly leading to neoplastic and metastatic transformation.



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Fig. 10. Sequence of signaling events during anoikis of primary human IECs. Loss of cell anchorage leads to the activation of long-prodomain caspase-2 and -9, followed by the rapid activation of downstream caspase-7, -3, and -6. Caspase 8 is also activated during IEC anoikis, presumably as a positive feedback mechanism during the augmentation phase of anoikis. Cytochrome c is released from mitochondria mostly during the augmentation phase of anoikis, possibly due to the cessation of FAK signaling via the phosphatidylinositol 3'-kinase (PI-3K)-Akt-Bad pathway. Apoptotic signaling eventually leads to DFF cleavage and DNA fragmentation, finalizing programmed cell death. Direct interaction of the components delineated needs to be shown in cell lines amenable to molecular/genetic manipulations.

 

    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
IEC Isolation and Induction of IEC Apoptosis.
Normal human intestinal mucosa from surgical specimens was obtained from patients undergoing surgery for large bowel neoplasia (>10 cm distant to pathology). IECs were isolated and purified as described previously (42) , with slight modification. Briefly, after dissection of the mucosa into small strips (3 x 20 mm) and mucus removal by 1 mM DTT in HBSS (30 min at ambient temperature), mucosal strips were incubated in 1 mM EDTA for 10 min at 37°C. Mucosal strips were rinsed briefly in HBSS and transferred to fresh HBSS, and IECs were detached by 10 shakes of the container. After quick removal of the strips (3 mm mesh), freshly detached intact IEC crypts were harvested in a nylon mesh (80 µm), washed out of the filter with medium (Keratinocyte Serum Free Medium; Life Technologies, Inc., Karlsruhe, Germany), and kept in suspension at 37°C as described previously (29) . Control experiments were performed with IECs isolated from human small bowel specimens yielding identical results.

To analyze IECs of different regions along the crypt-lumen axis in another set of experiments, sequential dispase incubations were undertaken as described previously (57) . Briefly, mucosal strips were incubated with dispase (1.2 units/ml) at 37°C. After 10, 20, and 30 min, IECs were extracted from the mucosal strip by vortex shaker (5 s), yielding epithelial cells of the upper, middle, and lower region of the intestinal crypt. Isolated IECs were washed in cold HBSS and resuspended in medium.

Flow Cytometry.
Because IECs are isolated as intact crypts using the protocol described above, single cell flow cytometry cannot be performed without further treatment of IECs to generate single cells. IECs harvested at indicated time points were therefore incubated for 2 min at 37°C with dispase (1.2 mg/ml; Boehringer Mannheim, Mannheim, Germany) followed by five vigorous shakes to fully disrupt cell-cell contacts. Enzyme activity was stopped by the addition of cold (4°C) EDTA (1 mM), and cells were washed twice in HBSS at 4°C. Cells were resuspended in 70% ice-cold methanol and stored at -20°C until analysis within 24 h. Flow cytometry analysis was performed according to standard technique (62) . Briefly, fixed cells were washed twice in PBS, treated with RNase (1.0 mg/ml; Boehringer Mannheim) for 30 min at 37°C, stained with 50 µg/ml PI (Sigma, St. Louis, MO), and kept in the dark on ice for 30 min before analysis. PI-stained cells were measured on a Coulter Elite XL cytometer (Beckman Coulter, Fullerton, CA) at 488 nm excitation. A 630 nm long pass filter was applied to the red fluorescence of PI.(FL3). A minimum of 5 x 103 cells were measured per sample, and gating was applied on the sidescatter count to exclude debris and on the pulse width count to exclude remaining cell aggregates from analysis. To assess purity, methanol-fixed cells were washed twice in PBS followed by blocking (20 min, PBS/2% FCS). Each batch of cells was incubated with isotype control (mouse IgG1-FITC; Coulter Immunotech Diagnostic, Hamburg, Germany), Ber-EP-4 (Dako Diagnostika GmbH, Hamburg, Germany), anti-CD3 (Coulter Immunotech), and anti-CD33 (Coulter Immunotech).

Caspase Activity Assay.
Caspase-3-like activity was determined using the synthetic tetrapeptide substrate N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD AMC; Biomol, Plymouth Meeting, PA) as described previously (63) . Briefly, 20 µg of IEC cytosol were incubated with 50 µM N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin for 30 min at 37°C, and release of fluorescence reflecting caspase-3-like activity was quantified by fluorometry as described previously (29) .

Western Blot.
IEC cytosol was extracted as described at different time points after loss of cell anchorage, and Western blot was performed as described previously (29) . The following primary antibodies were used: anti-caspase-9 (1:5000) and anti-caspase-2 (1:3000; both kindly provided by Sophie Roy; MerckFrost Pharmaceuticals, Quebec, Canada), anti-caspase-6 (1:3000; Becton Dickinson), DFF (1:1000; (Becton Dickinson). For Western blot studies on cytochrome c (anti-cytochrome c; 1:5000; Becton Dickinson), cells were lysed without sonication using digitonin as detergent as described previously (64) . Mitochondria were isolated as described after cell lysis, and purity of the mitochondrial cell fraction was confirmed by probing for the mitochondrial enzyme cytochrome c oxidase IV, subunit II [1:5000; Mobitec, Göttingen, Germany (64 , 65) ]. Equal loading of the cytosolic samples was demonstrated by reprobing membranes with anti-actin (1:10000; Chemicon, Hofheim, Germany).

Immunocytochemistry.
Ex vivo isolated human IECs were kept in suspension, and aliquots were taken at the indicated times. After cytospin (3 min at 300rpm), fixation and permeabilization were performed ("fix and perm" kit; An der Grube Bio Research GmbH, Kaumberg, Austria). Cells were then incubated with blocking buffer (2% BSA, 0.2% fat-free milk, 2% normal goat serum, and 0.8% Triton X-100 in PBS) followed by incubation with primary anti caspase-3 antibodies (CM-1 at 1:7500; kindly provided by IDUN Pharmaceuticals, La Jolla, CA) or isotope control for 1 h at ambient temperature. After washings in PBS, incubation with secondary biotinylated goat antirabbit antibody (Vectastain kit; Vector Laboratories, Burlingame, CA) for 1 h, and 1 h of incubation with avidin-biotin-peroxidase complex at ambient temperature, peroxidase substrate (3.3'-diaminobenzidine; Sigma) was added for 20 s, and the reaction was stopped by two washings in PBS.


    Acknowledgments
 
We thank the Department of Surgery, University of Regensburg for providing surgical specimens. We also thank Sophie Roy (MerckFrost Pharmaceuticals, Quebec, Canada) for kindly providing antibodies to caspase-2 and -9 and Anu Srinivasan (IDUN Pharmaceuticals, La Jolla, CA) for kindly providing anti-caspase-3 antibody (CM-1). Finally, we thank Alan D. Levine and Claudio Fiocchi (Case Western Reserve University, Cleveland, OH) for valuable advice and discussion.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by ReForM A and ReForM B (Medical Research Funding Program of the University of Regensburg, Regensburg, Germany) grants (to J. G.) and Grants GR1523/1-1, GR 1523/1-2, and GR1523/3-1 from the Deutsche Forschungsgemeinschaft, Bonn, Germany (to J. G.). Back

2 To whom requests for reprints should be addressed, at Department of Medicine I, University of Regensburg, Franz-Josef-Strasse-Allee 11, 93042 Regensburg, Germany. Phone: 49-941-944-7001; Fax: 49-941-944-7002; E-mail: Johannes.Grossmann{at}klinik.uni-regensburg.de Back

3 The abbreviations used are: IEC, intestinal epithelial cell; FAK, focal adhesion kinase; PI, propidium iodide; DFF, DNA fragmentation factor. Back

Received for publication 9/11/01. Revision received 1/ 8/01. Accepted for publication 1/ 8/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Potten C. S., Allen T. D. Ultrastructure of cell loss in intestinal mucosa. J. Ultrastr. Res., 60: 272-277, 1977.[Medline]
  2. Gavrieli Y., Sherman Y., Ben-Sasson S. A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol., 119: 493-501, 1992.[Abstract/Free Full Text]
  3. Sträter J., Koretz K., Gunthert A. R., Moller P. In situ detection of enterocytic apoptosis in normal colonic mucosa and in familial adenomatous polyposis. Gut, 37: 819-825, 1995.[Abstract/Free Full Text]
  4. Hall P. A., Coates P. J., Ansari B., Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J. Cell Sci., 107: 3569-3577, 1994.[Abstract/Free Full Text]
  5. Frisch S. M., Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol., 124: 619-626, 1994.[Abstract/Free Full Text]
  6. Beaulieu J. F. Differential expression of the VLA family of integrins along the crypt-villus axis in the human small intestine. J. Cell Sci., 102: 427-436, 1992.[Abstract/Free Full Text]
  7. Koretz K., Schlag P., Boumsell L., Moeller P. Expression of VLA-{alpha}2, VLA-{alpha}6, and VLA-ß1 chains in normal mucosa and adenomas of the colon, and in colon carcinomas and their liver metastases. Am. J. Pathol., 138: 741-750, 1991.[Medline]
  8. Zutter M. M., Santoro S. A. Widespread histologic distribution of the {alpha}2ß1 integrin cell-surface collagen receptor. Am. J. Pathol., 137: 113-120, 1990.[Medline]
  9. Probstmeier R., Martini R., Schachner M. Expression of J1/tenascin in the crypt-villus unit of adult mouse small intestine: implications for its role in epithelial cell shedding. Development (Camb.), 109: 313-321, 1990.[Abstract]
  10. Riedl S., Moller P., Faissner A., Schlag P. Induction and altered distribution of tenascin in the basal lamina of colorectal adenomas and carcinomas. Exs, 61: 277-281, 1992.[Medline]
  11. Gordon J. I., Hermiston M. L. Differentiation and self-renewal in the mouse gastrointestinal epithelium. Curr. Opin. Cell Biol., 6: 795-803, 1994.[Medline]
  12. Alnemri E. S., Livingston D. J., Nicholson D. W., Salvesen G., Thornberry N. A., Wong W. W., Yuan J. Human ICE/CED-3 protease nomenclature. Cell, 87: 171 1996.[Medline]
  13. Cohen G. M. Caspases: the executioners of apoptosis. Biochem. J., 326: 1-16, 1997.
  14. Stroh C., Schulze-Osthoff K. Death by a thousand cuts: an ever increasing list of caspase substrates. Cell Death Differ., 5: 997-1000, 1998.[Medline]
  15. Widmann C., Gibson S., Johnson G. L. Caspase-dependent cleavage of signaling proteins during apoptosis. J. Biol. Chem., 273: 7141-7147, 1998.[Abstract/Free Full Text]
  16. Nunez G., Benedict M. A., Hu Y., Inohara N. Caspases: the proteases of the apoptotic pathway. Oncogene, 17: 3237-3245, 1998.[Medline]
  17. Sun X. M., MacFarlane M., Zhuang J., Wolf B. B., Green D. R., Cohen G. M. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J. Biol. Chem., 274: 5053-5060, 1999.[Abstract/Free Full Text]
  18. Zhuang J., Cohen G. M. Release of mitochondrial cytochrome c is upstream of caspase activation in chemical-induced apoptosis in human monocytic tumour cells. Toxicol. Lett., 102–103: 121-129, 1998.
  19. King D., Pringle J. H., Hutchinson M., Cohen G. M. Processing/activation of caspases, -3 and -7 and -8 but not caspase-2, in the induction of apoptosis in B-chronic lymphocytic leukemia cells. Leukemia (Baltimore), 12: 1553-1560, 1998.[Medline]
  20. Slee E. A., Harte M. T., Kluck R. M., Wolf B. B., Casiano C. A., Newmeyer D. D., Wang H. G., Reed J. C., Nicholson D. W., Alnemri E. S., Green D. R., Martin S. J. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J. Cell Biol., 144: 281-292, 1999.[Abstract/Free Full Text]
  21. Li P., Nijhawan D., Budihardjo I., Srinivasula S. M., Ahmad M., Alnemri E. S., Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell, 91: 479-489, 1997.[Medline]
  22. Scaffidi C., Fulda S., Srinivasan A., Friesen C., Li F., Tomaselli K. J., Debatin K. M., Krammer P. H., Peter M. E. Two CD95 (APO-1/Fas) signaling pathways. EMBO J., 17: 1675-1687, 1998.[Abstract]
  23. Kuwana T., Smith J. J., Muzio M., Dixit V., Newmeyer D. D., Kornbluth S. Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c. J. Biol. Chem., 273: 16589-16594, 1998.[Abstract/Free Full Text]
  24. Meredith J. E., Jr., Fazeli B., Schwartz M. A. The extracellular matrix as a cell survival factor. Mol. Biol. Cell, 4: 953-961, 1993.[Abstract/Free Full Text]
  25. Boudreau N., Sympson C. J., Werb Z., Bissell M. J. Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science (Washington DC), 267: 891-893, 1995.[Abstract/Free Full Text]
  26. Werb Z., Sympson C. J., Alexander C. M., Thomasset N., Lund L. R., MacAuley A., Ashkenas J., Bissell M. J. Extracellular matrix remodeling and the regulation of epithelial-stromal interactions during differentiation and involution. Kidney Int., 54 (Suppl.): S68-S74, 1996.
  27. Vitale M., Di Matola T., Bifulco M., Casamassima A., Fenzi G., Rossi G. Apoptosis induced by denied adhesion to extracellular matrix (anoikis) in thyroid epithelial cells is p53 dependent but fails to correlate with modulation of p53 expression. FEBS Lett., 462: 57-60, 1999.[Medline]
  28. Sträter J., Wedding U., Barth T. F. E., Koretz K., Elsing C., Moller P. Rapid onset of apoptosis in vitro follows disruption of ß1-integrin/matrix interactions in human colonic crypt cells. Gastroenterology, 110: 1776-1784, 1996.[Medline]
  29. Grossmann J., Mohr S., Lapetina E. G., Fiocchi C., Levine A. D. Sequential and rapid activation of select caspases during apoptosis of normal intestinal epithelial cells. Am. J. Physiol., 274: G1117-G1124, 1998.[Abstract/Free Full Text]
  30. Haake A. R., Polakowska R. R. Cell death by apoptosis in epidermal biology. J. Investig. Dermatol., 101: 107-112, 1993.[Medline]
  31. Tamada Y., Takama H., Kitamura T., Yokochi K., Nitta Y., Ikeya T., Matsumoto Y. Identification of programmed cell death in normal human skin tissues by using specific labelling of fragmented DNA. Br. J. Dermatol., 131: 521-524, 1994.[Medline]
  32. Re F., Zanetti A., Sironi M., Polentarutti N., Lanfrancone L., Dejana E., Colotta F. Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J. Cell Biol., 127: 537-546, 1994.[Abstract/Free Full Text]
  33. Aoshiba K., Rennard S. I., Spurzem J. R. Cell-matrix and cell-cell interactions modulate apoptosis of bronchial epithelial cells. Am. J. Physiol., 272: L28-L37, 1997.[Abstract/Free Full Text]
  34. Kornberg L. J. Focal adhesion kinase and its potential involvement in tumor invasion and metastasis. Head Neck, 20: 745-752, 1998.[Medline]
  35. Ruoslahti E., Reed J. C. Anchorage dependence, integrins, and apoptosis. Cell, 77: 477-478, 1994.[Medline]
  36. Guan J. L., Shalloway D. Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Nature (Lond.), 358: 690-692, 1992.[Medline]
  37. Shaw L. M., Rabinovitz I., Wang H. H., Toker A., Mercurio A. M. Activation of phosphoinositide 3-OH kinase by the {alpha}6ß4 integrin promotes carcinoma invasion. Cell, 91: 949-960, 1997.[Medline]
  38. van de Water B., Nagelkerke J. F., Stevens J. L. Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells. J. Biol. Chem., 274: 13328-13337, 1999.[Abstract/Free Full Text]
  39. Hermiston M. L., Gordon J. I. In vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death. J. Cell Biol., 129: 489-506, 1995.[Abstract/Free Full Text]
  40. McGill G., Shimamura A., Bates R. C., Savage R. E., Fisher D. E. Loss of matrix adhesion triggers rapid transformation-selective apoptosis in fibroblasts. J. Cell Biol., 138: 901-911, 1997.[Abstract/Free Full Text]
  41. Fukai F., Mashimo M., Akiyama K., Goto T., Tanuma S., Katayama T. Modulation of apoptotic cell death by extracellular matrix proteins and a fibronectin-derived antiadhesive peptide. Exp. Cell Res., 242: 92-99, 1998.[Medline]
  42. Grossmann J., Maxson J. M., Whitacre C. M., Orosz D. E., Berger N. A., Fiocchi C., Levine A. D. New isolation technique to study apoptosis in human intestinal epithelial cells. Am. J. Pathol., 153: 53-62, 1998.[Medline]
  43. Frisch S. M., Vuori K., Ruoslahti E., Chan-Hui P. Y. Control of adhesion-dependent cell survival by focal adhesion kinase. J. Cell Biol., 134: 793-799, 1996.[Abstract/Free Full Text]
  44. Levkau B., Herren B., Koyama H., Ross R., Raines E. W. Caspase-mediated cleavage of focal adhesion kinase pp125FAK and disassembly of focal adhesions in human endothelial cell apoptosis. J. Exp. Med., 187: 579-586, 1998.[Abstract/Free Full Text]
  45. Cardone M. H., Roy N., Stennicke H. R., Salvesen G. S., Franke T. F., Stanbridge E., Frisch S., Reed J. C. Regulation of cell death protease caspase-9 by phosphorylation. Science (Washington DC), 282: 1318-1321, 1998.[Abstract/Free Full Text]
  46. Khwaja A., Rodriguez-Viciana P., Wennstrom S., Warne P. H., Downward J. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J., 16: 2783-2793, 1997.[Abstract]
  47. Takeichi M. Cadherins in cancer: implications for invasion and metastasis. Curr. Opin. Cell Biol., 5: 806-811, 1993.[Medline]
  48. Kinch M. S., Petch L., Zhong C., Burridge K. E-cadherin engagement stimulates tyrosine phosphorylation. Cell Adhes. Commun., 4: 425-437, 1997.[Medline]
  49. Behrens J. Cadherins and catenins: role in signal transduction and tumor progression. Cancer Metastasis Rev., 18: 15-30, 1999.[Medline]
  50. Cardone M. H., Salvesen G. S., Widmann C., Johnson G., Frisch S. M. The regulation of anoikis: MEKK-1 activation requires cleavage by caspases. Cell, 90: 315-323, 1997.[Medline]
  51. Frisch S. M., Vuori K., Kelaita D., Sicks S. A role for Jun-N-terminal kinase in anoikis; suppression by bcl-2 and crmA. J. Cell Biol., 135: 1377-1382, 1996.[Abstract/Free Full Text]
  52. Khwaja A., Downward J. Lack of correlation between activation of Jun-NH2-terminal kinase and induction of apoptosis after detachment of epithelial cells. J. Cell Biol., 139: 1017-1023, 1997.[Abstract/Free Full Text]
  53. Krestow J. K., Rak J., Filmus J., Kerbel R. S. Functional dissociation of anoikis-like cell death and activity of stress activated protein kinase. Biochem. Biophys. Res. Commun., 260: 48-53, 1999.[Medline]
  54. Frisch S. M. Evidence for a function of death-receptor-related, death-domain-containing proteins in anoikis. Curr. Biol., 9: 1047-1049, 1999.[Medline]
  55. Rytomaa M., Martins L. M., Downward J. Involvement of FADD and caspase-8 signalling in detachment-induced apoptosis. Curr. Biol., 9: 1043-1046, 1999.[Medline]
  56. Grossmann J., Artinger M., Grasso A. W., Kung H. J., Scholmerich J., Fiocchi C., Levine A. D. Hierarchical cleavage of focal adhesion kinase by caspases alters signal transduction during apoptosis of intestinal epithelial cells. Gastroenterology, 120: 79-88, 2001.[Medline]
  57. Mayer L., Panja A., Li Y., Siden E., Pizzimenti A., Gerardi F., Chandswang N. Unique features of antigen presentation in the intestine. Ann. N. Y. Acad. Sci., 664: 39-46, 1992.[Medline]
  58. Liu X., Zou H., Slaughter C., Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell, 89: 175-184, 1997.[Medline]
  59. Ilic D., Almeida E. A., Schlaepfer D. D., Dazin P., Aizawa S., Damsky C. H. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J. Cell Biol., 143: 547-560, 1998.[Abstract/Free Full Text]
  60. Muzio M., Stockwell B. R., Stennicke H. R., Salvesen G. S., Dixit V. M. An induced proximity model for caspase-8 activation. J. Biol. Chem., 273: 2926-2930, 1998.[Abstract/Free Full Text]
  61. Chandler J. M., Cohen G. M., MacFarlane M. Different subcellular distribution of caspase-3 and caspase-7 following Fas-induced apoptosis in mouse liver. J. Biol. Chem., 273: 10815-10818, 1998.[Abstract/Free Full Text]
  62. Darzynkiewicz Z., Bruno S., Del Bino G., Gorczyca W., Hotz M. A., Lassota P., Traganos F. Features of apoptotic cells measured by flow cytometry. Cytometry, 13: 795-808, 1992.[Medline]
  63. Thornberry N. A., Bull H. G., Calaycay J. R., Chapman K. T., Howard A. D., Kostura M. J., Miller D. K., Molineaux S. M., Weidner J. R., Aunins J., et al A novel heterodimeric cysteine protease is required for interleukin-1ß processing in monocytes. Nature (Lond.), 356: 768-774, 1992.[Medline]
  64. Leist M., Volbracht C., Fava E., Nicotera P. 1-Methyl-4-phenylpyridinium induces autocrine excitotoxicity, protease activation, and neuronal apoptosis. Mol. Pharmacol., 54: 789-801, 1998.[Abstract/Free Full Text]
  65. Bossy-Wetzel E., Newmeyer D. D., Green D. R. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J., 17: 37-49, 1998.[Abstract]



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