Expression of the Vacuolar H+-ATPase 16-kDa Subunit Results in the Triton X-100-insoluble Aggregation of β1 Integrin and Reduction of Its Cell Surface Expression

Vacuolar H+-ATPase functions as a vacuolar proton pump and is responsible for acidification of intracellular compartments such as the endoplasmic reticulum, Golgi, lysosomes, and endosomes. Previous reports have demonstrated that a 16-kDa subunit (16K) of vacuolar H+-ATPase via one of its transmembrane domains, TMD4, strongly associates with β1 integrin, affecting β1 integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of β1 integrin-mediated HEK-293 cell motility by 16K expression, we investigated the mechanism by which 16 kDa was having this effect. Using HT1080 cells whose α5β1 integrin-mediated adhesion to fibronectin has been extensively studied, the expression of 16 kDa also resulted in reduced cell spreading on fibronectin-coated substrates. A pulse-chase study of β1 integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of β1 integrin (premature form) and, consequently, the level of the 130-kDa form of β1 integrin (mature form). Further experiments showed that the normal levels of association between the premature β1 integrin form and calnexin were significantly decreased by the expression of either 16 kDa or TMD4. Expression of 16 kDa also resulted in a Triton X-100-insoluble aggregation of an unusual 87-kDa form of β1 integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits β1 integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional β1 integrin. Vacuolar H+-ATPase functions as a vacuolar proton pump and is responsible for acidification of intracellular compartments such as the endoplasmic reticulum, Golgi, lysosomes, and endosomes. Previous reports have demonstrated that a 16-kDa subunit (16K) of vacuolar H+-ATPase via one of its transmembrane domains, TMD4, strongly associates with β1 integrin, affecting β1 integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of β1 integrin-mediated HEK-293 cell motility by 16K expression, we investigated the mechanism by which 16 kDa was having this effect. Using HT1080 cells whose α5β1 integrin-mediated adhesion to fibronectin has been extensively studied, the expression of 16 kDa also resulted in reduced cell spreading on fibronectin-coated substrates. A pulse-chase study of β1 integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of β1 integrin (premature form) and, consequently, the level of the 130-kDa form of β1 integrin (mature form). Further experiments showed that the normal levels of association between the premature β1 integrin form and calnexin were significantly decreased by the expression of either 16 kDa or TMD4. Expression of 16 kDa also resulted in a Triton X-100-insoluble aggregation of an unusual 87-kDa form of β1 integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits β1 integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional β1 integrin. Vacuolar H+-ATPase (V-ATPase) 1The abbreviations used are: V-ATPase, vacuolar H+-ATPase; ConA, concanavalin A; 16K, 16-kDa subunit; DMEM, Dulbecco's modified Eagle's medium; HSV, herpes simplex virus; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; TMD4, transmembrane domain 4; ERK, extracellular signal-regulated kinase; HRP, horseradish peroxidase; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Endo H, endoglycosidase H; pHi, intracellular pH. is a multi-subunit complex found in all of the eukaryotic cells and consists of the V1 domain, which contains the ATPase activity, and the V0 domain, which contains transmembrane proton channel. V-ATPase is ubiquitously expressed in the various intracellular compartments and is responsible for their acidification (1Arai H. Terres G. Pink S. Forgac M. J. Biol. Chem. 1988; 263: 8796-8802Abstract Full Text PDF PubMed Google Scholar, 2Forgac M. Ann. N. Y. Acad. Sci. 1992; 671: 273-283Crossref PubMed Scopus (9) Google Scholar). The 16-kDa subunit (16K) of the V0 domain is a highly hydrophobic protein, consisting of four transmembrane segments, and it forms a homohexamer that functions as the transmembrane proton channel. The 16K can associate with other proteins through interactions with its fourth transmembrane segment, TMD4. For example, the papillomavirus E5 oncogene product associates with the TMD4 of 16K, decoupling 16 kDa from the ATPase V1 and resulting in an elevated pH in the Golgi and facilitation of the oncogenic transformation process. In addition, the complex between bovine papillomavirus E5 and the 16K can associate with the platelet-derived growth factor receptor, leading to a ligand-independent activation of platelet-derived growth factor receptor signaling (3Goldstein D.J. Andresson T. Sparkowski J.J. Schlegel R. EMBO J. 1992; 11: 4851-4859Crossref PubMed Scopus (116) Google Scholar). β1 integrin performs a critical function in the anchorage-dependent growth of many cell types and is transcriptionally regulated by a variety of factors during development and oncogenic transformation (4Vihinen P. Nikkola J. Vlaykova T. Hahka-Kemppinen M. Talve L. Heino J. Pyrhonen S. Melanoma Res. 2000; 10: 243-251Crossref PubMed Scopus (19) Google Scholar, 5Perlino E. Lovecchio M. Vacca R.A. Fornaro M. Moro L. Ditonno P. Battaglia M. Selvaggi F.P. Mastropasqua M.G. Bufo P. Languino L.R. Am. J. Pathol. 2000; 157: 1727-1734Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 6Milner R. Campbell I.L. Mol. Cell Neurosci. 2002; 20: 616-626Crossref PubMed Scopus (109) Google Scholar, 7Heino J. Ignotz R.A. Hemler M.E. Crouse C. Massague J. J. Biol. Chem. 1989; 264: 380-388Abstract Full Text PDF PubMed Google Scholar, 8Delcommenne M. Streuli C.H. J. Biol. Chem. 1995; 270: 26794-26801Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Plath T. Detjen K. Welzel M. von Marschall Z. Murphy D. Schirner M. Wiedenmann B. Rosewicz S. J. Cell Biol. 2000; 150: 1467-1478Crossref PubMed Scopus (109) Google Scholar). The TMD4 of 16 kDa has also been shown to mediate an association between 16K and β1 integrin (10Skinner M.A. Wildeman A.G. J. Biol. Chem. 1999; 274: 23119-23127Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Subsequently, it was shown that the 16K expression in HEK-293 cells caused altered N-linked glycosylation of β1 integrin, resulting in a significant inhibition of cell migration toward laminin and fibronectin (10Skinner M.A. Wildeman A.G. J. Biol. Chem. 1999; 274: 23119-23127Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 11Skinner M.A. Wildeman A.G. J. Biol. Chem. 2001; 276: 48451-48457Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Since altered expression of N-linked glycans on the α5β1 integrin has been shown to significantly affect the binding of cell surface α5β1 to fibronectin, it was reasonable to hypothesize that the expression of 16 kDa had specific effects on the glycosylation of β1 integrin, thereby inhibiting its adhesive functions that regulate migration on extracellular matrix. In this study, we investigated the mechanism of how the 16K expression levels affect the expression and/or the maturation and glycosylation of β1 integrin during the biosynthesis of the α5β1 dimer to ultimately inhibit β1 integrin-dependent cell adhesion to fibronectin. The results showed that expression of the 16 kDa in human fibrosarcoma HT1080 cells down-regulates surface expression of α5β1 integrin dimers, thereby causing reduced cell spreading on fibronectin-coated surfaces. The loss of cell surface α5β1 expression was subsequently shown to result from a Triton X-100-insoluble aggregation of β1 integrin subunits in the endoplasmic reticulum. Initial results suggested that the 16K expression suppressed the maturation of β1 integrin from its premature form that expressed only high-mannose and not complex glycans. Pulse-chase experiments then showed that premature (110 kDa) β1 integrin levels in 16K-transfected cells were greatly decreased and that this decrease occurred during the first 15 min of β1 integrin biosynthesis. Further experiments using antibodies against calnexin, calreticulin, and BiP showed that calnexin, but not calreticulin or BiP, associated with the premature β1 integrin. When 16K was expressed, the normal association between the premature β1 integrin and calnexin was almost completely abolished. The expression of the TMD4 alone caused the same level of inhibition on β1 integrin association with calnexin. Surprisingly, both 16K and TMD4 were themselves co-immunoprecipitated with calnexin. An examination of the Triton X-100-insoluble fraction of cells transfected with 16K revealed a new form of β1 with a molecular mass of ∼87 kDa. The unusually rapid kinetics of the decrease of the premature (110 kDa) β1 integrin and the appearance of a Triton X-100-insoluble 87-kDa form of β1 suggest that the decrease of the 110-kDa form was not due primarily to ER-associated degradation (ERAD) but to an induction of the Triton X-100-insoluble form of β1. These results suggested a unique mechanism for the regulation of the cell surface expression of β1 integrin by expression of the V-ATPase 16-kDa subunit. Cell Culture, Transfections, and Materials—HT1080 human fibrosarcoma cells were obtained from ATCC and maintained in DMEM containing 10% fetal bovine serum. Unless otherwise described, all of the transfections were done transiently using Lipofectamine reagent (Invitrogen) in 100-mm dishes. Cells were harvested typically between 24 and 48 h after transfection and used for further experiments. Rat monoclonal antibodies against human α5 and β1 integrins, mAb16 and mAb13, respectively, were gifts from Dr. Steven Akiyama (NIEHS, National Institutes of Health). The anti-T7 and HSV-tagged antibodies were purchased from Novagen. Antibodies against calnexin, calreticulin, and BiP were purchased from Stressgen. Agarose-conjugated ConA, and were obtained from an of activity, was from was obtained from and was from was purchased from and all of the antibodies to were from and were all purchased from expression and bovine β1 were obtained as M.A. Wildeman A.G. J. Biol. Chem. 2001; 276: 48451-48457Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). and Western were in a consisting of Triton and of was used for and were with otherwise to in bovine and with with antibodies or proteins were by the reagent of the was using a Cell and were with as M. Akiyama M. Res. 2002; Google Scholar). Cells were with in dishes. in was to the cells and for 15 Cells were then in and in and Western blotting were done as with the that was with and were with DMEM (Invitrogen) for min by with in of DMEM and but containing fetal bovine for 15 the growth was and the cells were for various cells were then in and cell were with either or anti-T7 For an experiment with of in was to the cells during both and were transfected with alone or with of and for 24 The was used to was done using and using the and were used for and were using of used in the experiment were as human α5 integrin and human β1 integrin and human 16K and and human and was using the The were for by for for for and for with for for for human α5 and β1 integrin were by a of obtained from the and were used for for 16K and GAPDH, were with fibronectin and transfected cells were harvested by and in in DMEM for h cells were to and to on for Cells were then with in for 15 with PBS, with Triton in for 15 and with bovine for min Cells were then with and for with PBS, the antibodies were by with either or for For was with PBS, the were and were obtained using an Expression of the V-ATPase 16K in HEK-293 cells significantly β1 integrin-mediated cell adhesion and migration on fibronectin and laminin M.A. Wildeman A.G. J. Biol. Chem. 2001; 276: 48451-48457Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In addition, the expression of 16K to have an on the complex N-linked glycosylation of β1 integrin, that the inhibition of matrix adhesion have resulted from altered glycosylation of β1 integrin. this in we used HT1080 cells to the effects of 16K expression on β1 interactions as as the biosynthesis of β1. The β1 integrin expressed in HT1080 cells is α5β1 integrin M. Akiyama M. Res. 2002; Google and cells have been used to the effects of altered N-linked glycosylation on α5β1 integrin-mediated cell adhesion and migration on fibronectin M. Akiyama M. Res. 2002; Google Scholar, J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). 16K Expression Cell and Cell Expression of α5β1 we that 16K expression α5β1 integrin-mediated HT1080 cell adhesion and spreading on fibronectin. HT1080 cells were transiently transfected with 16K with the in in DMEM for to fibronectin-coated and to for was used to the of the and was used to 16K The results showed that cells transfected with alone showed spreading on the fibronectin cells transfected with were to the but were in appearance due to the inhibition of were then to the effects of 16K expression on β1 integrin cell surface expression using cell surface The results showed a decrease of α5β1 integrin expression on the cell surface as the of 16K used for transfection was Cell surface expression of both the growth factor receptor and showed significant decrease after transfection with 16K a specific on α5β1 integrin expression down-regulates surface expression of α5β1 integrin in surface expression of α5 β1 integrin by 16K expression is Cells were transiently transfected with of 16K expression 24 cell surface proteins were using and in a containing Triton Cell were with β1 integrin, mAb13, by and with The on the levels of surface expression of the growth factor receptor on cells transfected with alone or 15 of cell surface were 16K was Western of and of β1 a first to the effects of 16K expression on β1 integrin cell surface expression, it was to its biosynthesis. β1 integrin is first expressed as the (110 which with subunits and is to the Golgi complex it then the 130-kDa form that complex N-linked glycans J. Ignotz R.A. Hemler M.E. Crouse C. Massague J. J. Biol. Chem. 1989; 264: 380-388Abstract Full Text PDF PubMed Google Scholar, D. M. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, P. Heino J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The premature and forms can also on Western by the binding of specific ConA, which and both the premature and forms of and S. which and respectively, only to the β1 When bovine β1 integrin was expressed in HT1080 both the and premature forms of the integrin were by Western blotting of cell The levels of the premature form were due to levels of integrin subunits to with which is a for Golgi and complex N-linked also showed that only the kDa) form of β1 was with a mAb16 against human α5 integrin and the complex N-linked and S. but the premature (110 kDa) form was only by of β1 by 16K HSV-tagged 16K was with the expression of the β1 form was of the of cells transfected with β1 integrin or β1 integrin 16K with the specific for the α5 subunit revealed that the β1 integrin co-immunoprecipitated with α5 integrin was significantly decreased after 16K expression 16K expression suppressed the expression of the premature form as the of 16K used for transfections was the of The results showed that the levels of the premature β1 form were greatly decreased after 16K The levels of α5 integrin and showed significant decrease as the expression of 16K that the on β1 expression was specific When cell from cells with β1 integrin and were with β1 co-immunoprecipitated with the that 16K associated with β1 integrin. and Western the β1 that co-immunoprecipitated with 16K to β1 integrin with Endo not the premature β1 integrin form not for h and maturation the 130-kDa form 16K expression had effects on the β1 integrin expression and maturation as it had on we the biosynthesis of the β1 using and an integrin A pulse-chase experiment with cells transfected with 16K revealed that was a significant decrease in premature β1 integrin levels as resulting in an level of α5β1 integrin after h of the the decrease of expressed β1 integrin we pulse-chase study with and In premature β1 integrin levels were down-regulated by the In cells transfected with was significant of premature β1 integrin level during the and that the of the premature β1 decrease caused by 16K expression occurred during the Because the as a for or subunits of Cell Biol. 2002; PubMed Scopus Google Scholar, L. Mol. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google we the decrease of β1 integrin levels is due to degradation by the using a The result showed that the decrease of β1 integrin levels is not by the the of both and resulted in a level of premature β1 integrin. this result and the unusually rapid kinetics of the decrease of premature β1 integrin levels 15 suggested that the decreased β1 integrin level was due to degradation by a The β1 from 16K Expression to premature β1 integrin levels after 16K expression caused by a decrease in the of β1 integrin, we 16K expression of either α5 or β1 integrin using was significant in the levels of either α5 or β1 integrin that the decrease in premature β1 integrin levels expression of 16K was not due to of β1 integrin. Expression of either 16K or the TMD4 of 16K of the β1 with the biosynthesis of in the ER, and was by various and calnexin, calreticulin, and BiP PubMed Scopus Google Scholar, M. 2000; PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar). integrin and β1 premature forms associate with calnexin, which is to result in a of integrin subunits J. Cell Biol. 1995; PubMed Scopus Google Scholar, M. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of β1 integrin with was to 16K expression interactions during of premature β1 integrin in the Interestingly, of β1 integrin and 16K caused a decrease in the association of the premature β1 integrin with calnexin, but was association with calreticulin or BiP either or after expression of 16K Because of the function of 16K as a of V-ATPase, we that an intracellular pH resulting from expression of the proton responsible for the effects on the decreased association between calnexin and the premature β1. as one to this TMD4 as the domain of 16 kDa that β1 integrin (10Skinner M.A. Wildeman A.G. J. Biol. Chem. 1999; 274: 23119-23127Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 11Skinner M.A. Wildeman A.G. J. Biol. Chem. 2001; 276: 48451-48457Abstract Full Text Full Text PDF PubMed Scopus (17) Google we expressed the HSV-tagged TMD4 of 16 kDa M.A. Wildeman A.G. J. Biol. Chem. 2001; 276: 48451-48457Abstract Full Text Full Text PDF PubMed Scopus (17) Google to TMD4 alone the 16K and the same level of inhibition on calnexin association with premature β1 The expression of TMD4 alone was to all of the effects of 16K expression on β1 integrin inhibition of premature β1 subunit association with calnexin 16K expression TMD4 expression showed an on α5 integrin association with calnexin, the of its with the β1 subunit. with 16K or TMD4 for the inhibition of between the premature β1 subunit and calnexin by 16K was that calnexin also associate with 16K. The results of experiments to this that both 16K and TMD4 were co-immunoprecipitated with calnexin an association between 16K and calnexin. result was obtained both by of of cells 16K with by Western blotting with and by by Western blotting with further this a pulse-chase study with both and cells was using an and, in both a was was co-immunoprecipitated with 16K after h of TMD4 was co-immunoprecipitated with this after Expression of 16K a Triton X-100-insoluble of an 87-kDa of β1 results a the of the β1 integrin forms that were not with calnexin after 16K Because the decrease in premature β1 integrin levels was not caused by or ER-associated degradation and and calnexin association with we that an association of premature β1 integrin with 16K in the result in an aggregation of the premature β1 subunits that then from the Triton fraction of cell used for this cells transfected with alone or were in Triton and, after of for of proteins from both were as The Triton were to the Triton X-100-insoluble were in or and to by Western blotting with anti-T7 The results showed that 16K was expressed, a form of β1 integrin of molecular mass kDa) was in the Triton X-100-insoluble but not in the Triton fraction result suggested that the of β1 subunits in the fraction of cell after 16K expression was due to an in β1 subunit which in the Triton X-100-insoluble The 87-kDa form was after the in either or that the aggregation was not due to α5 integrin the protein, showed of this form of aggregation and in Triton the 87-kDa form of β1 was not with that the of the molecular mass of this form was due to N-linked both of the molecular mass forms were with the anti-T7 In results have demonstrated that the V-ATPase 16K can regulate surface expression of β1 integrin, on biosynthesis thereby inhibits β1 integrin-mediated cell spreading on fibronectin. These were to the mechanism by which the expression of the 16-kDa subunit of the vacuolar ATPase to have a specific on the expression of complex on β1 integrin, which in have demonstrated that expression on β1 integrin subunits is for interactions J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, T. J. R. Y. M. E. K. K. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of that during oncogenic transformation results in the of integrin and cell M. Akiyama M. Res. 2002; Google Scholar, T. J. R. Y. M. E. K. K. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. S. J. Cell Biol. 1995; PubMed Scopus Google Scholar). the effects of 16K expression on β1 integrin biosynthesis were which to the of the that an association between the 16K and β1 integrin in the in the regulation of β1 subunit maturation and surface during the of this study, we a novel between calnexin and 16K. The V-ATPase proton of a of of the 16-kDa subunit of four transmembrane of which of highly hydrophobic 16K were to in the with typically from to in to of the TMD4 is unique the four of it was by to the V-ATPase to on the of the proton it with transmembrane The used in experiments of between the and the fourth and of the fourth transmembrane domain by of the on a T. S. Forgac M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google the the fourth transmembrane domain was found on the the was toward the of only that in the of TMD4 with either the domain or of calnexin. In this study, it is calnexin associates with 16K in the or other proteins in this calnexin to toward highly hydrophobic transmembrane E. M. C. J. PubMed Scopus Google Scholar, Y. K. M. K. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google E. S. P. EMBO J. PubMed Scopus Google and P. EMBO J. 2001; 20: PubMed Scopus Google a of calnexin in the of transmembrane domain These with the that calnexin with 16K to function in the of 16K biosynthesis. Because one of the functions of calnexin is to for and by the of 16K expression on the between calnexin and β1 integrin the Triton X-100-insoluble aggregation of the β1 subunit. is that the binding of 16K to premature β1 integrin the of the β1 subunit that it can associate with calnexin, leading to aggregation of β1 integrin in the A of inhibition of α5 by 16K for the in which the binding of 16K to the β1 subunit is a for the inhibition of integrin association with calnexin in the The Triton X-100-insoluble β1 integrin that resulted from 16K expression a molecular mass kDa) that of the premature β1 form (110 and it was not by in to the premature The for the of the 87-kDa form to by is the has been only to in the C. D. EMBO J. PubMed Scopus Google Scholar). C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that the regulated β1 integrin maturation and surface Using demonstrated that the maturation of β1 integrin and its surface expression, resulting in the of premature β1 integrin in the results demonstrated an of a protein, 16K, whose expression can regulate surface expression of β1 integrin and β1 integrin-mediated cell interactions with the extracellular matrix. from 16K function in β1 integrin biosynthesis and surface a functional between a in vacuolar proton and its association with a cell adhesion such as β1 Interestingly, a between that intracellular the and the of interactions has been M.A. G. C. Acad. Sci. S. 1989; PubMed Scopus Google demonstrated that many anchorage-dependent cells showed an of their intracellular pH cell spreading on cells with or showed to M.A. G. C. Acad. Sci. S. 1989; PubMed Scopus Google Scholar). have that cell spreading on extracellular matrix caused a that was shown to by the activation of via various and and that cell spreading was for M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. M.A. R. PubMed Scopus Google Scholar). have revealed that the subunit of the V1 ATPase domain associated with resulting in of the subunit of the V1 domain the binding of the V-ATPase to the of effects to cell spreading and K. S. S. J. K. H. T. K. J. T. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, H. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the of V-ATPase in various of In of with the of the TMD4 of 16K to cell growth T. Sparkowski J. D.J. Schlegel R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google results suggested that factors in the regulation of cell spreading of anchorage-dependent cells and that regulation of both and vacuolar an for loss of the anchorage-dependent and for and critical of the also Dr. for the of to Steven and Steven Akiyama for and this

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