[CANCER RESEARCH 55, 354-359, January 15, 1995J

Transgenic mice homozygously lacking in the bcl-2 gene were generatedusing homologous recombination in embryonal stem ceHs. The complete

absence of Bcl-2a and -@ proteins did not interfere with normal embry

onic development. Abnormalities became evident after birth, although theseverity varied among homozygous null mice. bcl-2@ mice displayedplelotropic abnormalities similar to those in the previously described

bcl-2' mice, including growth retardation, smaller ears, short lives,polycystic kidney, atrophic thymus and spleen with accelerated apoptoticcell death of lymphocytes, and hair hypopiginentation in the second hairfollicle cycle. Our bcl-2' mice also revealed novel defects in the smallintestine, characterized by retarded development, accelerated exfoliationof epitheial cells, and very few mitotic progenitor cells.

INTRODUCTION

The bcl-2 proto-oncogene was identified originally through studyof the t(14;18) translocation present in human B cell follicular lymphomas (1—3).This translocation juxtaposes the bcl-2 proto-oncogeneto the immunoglobulin heavy-chain gene locus, resulting in deregulation of bcl-2 gene expression (1). bcl-2 is unique among cellulargene products in its ability to enhance lymphoid cell survival byinterfering with apoptotic cell death rather than by promoting cellproliferation (4—8).Neuronal cell death induced by deprivation ofneurotrophic factors also is inhibited by the Bcl-2 protein (9, 10).Bcl-2 protein has been reported to be present in multiple membranelocations, including nuclear outer membrane, endoplasmic reticulummembrane, and mitochondrial outer membrane (7, 11—15).

The bcl-2 gene is expressed in a variety of tissues and cell types inembryos and adults, although expression is more restricted in adults(16,17).Severalbiologicalfunctionsofbcl-2havebeenconsidered,such as selection of lymphoid cells; determination of the life span ofcells such as lymphoid progenitors, neurons, and epithelial progenitors in intestine and epidermis; and possibly some role in differentiation and morphogenesis (17).

Cene targeting in ES3 cells has proven extremely useful for thestudy of gene functions (18). Mice homologously lacking in the bcl-2gene have been reported to show a variety of abnormalities (19, 20).We independently generated the bcl-2-deficient mouse and observednot only abnormalities similar to those described previously but alsoadditional defects in the small intestine.

Received 7/8/94; accepted 11/10/94.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported in part by a Grant-in-Aid for the comprehensive 10-Year Strategy for

Cancer Control from the Ministry of Health and Welfare, for Scientific Research onPriority Areas, and for Scientific Research from the Ministry of Education, Science andCulture of Japan, and Uehara Memorial Foundation; and by a grant from OsakaUniversity.

2 To whom requests for reprints should be addressed.

3 The abbreviation used is: ES, embryonic stem.

Construction of Targeting Vector. A genomic clone containing the 5'-end of the bcl-2 gene was obtained from the D3 ES cell (21) genomic library.The DNA segment containing the first and second exons of the bc!-2 gene wasused to prepare the targeting construct, pHd (Fig. IA). The short arm of pBcl

was a 1.4-kilobase ApaI-Kpnl fragment (nucleotides 360—1790; cf Ref. 22)from the region upstream of the coding exon II; this fragment contains thenoncoding exon I. The long arm was a 7.5-kilobase BamHI (nucleotide 2374;cj: Ref. 22)-Sau3AI fragment from the region downstream of the coding exon

II; this fragment contains 7 of the 192 amino acids in the second exon.Additional fragments used to construct the targeting vector included a 1.8-kilobase EcoRI-HindIII fragment from pGEM7(Neo) containing the PGK-neo

polyadenylated cassette conferring 0418 resistance under the control of thephosphoglycerate kinase promoter (23), and a 2.4-kilobase EcoRI-HindIllfragment from pGEM7@I'K) containing the PGK-thymidine kinase-polyadenylated cassette. The pBcl vector was constructed such that almost the entirecoding exon II was replaced by the PGK-neo-polyadenylated fragment; therefore, neither Bcl-2a nor Bcl-2@protein is generated.

Transfectlon and Selection of Mutant ES Cells. The E14 ES cell line(24) derived from a 129/Ola male mouse blastocyst was grown on NHL7

feeder cells as described (25). The targeting vector pBcl was linearized withSal! and electroporatedinto E14 cells in HEPES-buffered saline at 25 @xgofDNA/b7 cells. After 48 h, cells were selected with 0418 (400 @xWml).Cellswith a disrupted bcl-2 gene were initially screened by PCR and confirmed by

Southern analysis, using a 1.8-kilobase EcoRI-HindllI fragment located upstream of the 5'-end of the short arm of pBcl (Fig. IA).

Generation of Germilne Chimeras. Blastocysts were recovered from thematings of BCF1 (C57BL16 X C3H F1, hereafter called B6C3F1) and C57BL/6mice. ES-injected blastocysts were reimplanted into pseudopregnant ICR mice.Chimeric mice were mated with ICR mice. Because of the genotypes determining coat color of 129/Ola (Aw/Aw,b/b, c@/c'@',pip) and ICR (c/c) mice,germ line transmission of the ES-derived genotype is recognizable by the lightchinchilla coat color of the mouse. Offspring were screened by Southernanalysis. Heterozygous mice were interbred to produce homozygous offspring.

Western Blot Analysis. Frozen tissues were homogenized and subse

quently lysed in lysis buffer [50 mM Tris-HCI (pH 7.5), 150 mM NaG, 0.1%SDS, 1%NP4O,0.1%sodiumdeoxycholate,10 ,xMp-amidinophenylmethanesulfonyl fluoride hydrochloride, 0.1 @Wmiaprotinin, 5 p@@mlleupeptin, 10mM sodium diphosphate decahydrate, 10 mM NaF, 4 mM EDTA, and 2 mMNa3VO4].Protein samples (30 @geach) were separated by SDS-polyacrylamide (12%) gel electrophoresis and blotted onto Immobion polyvinylidenedifluoride membrane (Millipore). Bcl-2 was detected using anti-glutathioneS-transferase mouse Bcl-2@3polyclonal rabbit antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG, and enhanced chemiluminescence mixture detection reagent (Amersham).

Flow Cytometry. Single-cell suspensions were prepared from thymus andspleen. Aliquots of 106 cells were placed in individual wells of 96-wellmicrotiter plates and were incubated with optimally diluted antibodies in PBScontaining 1% fetal bovine serum for 30 mm at 4°C.FITC-conjugated antiCD4, phycoerythrin-conjugated anti-CD8, FITC-conjugated anti-IgM, andphycoerythrin-conjugated B220 were purchased from Pharmingen. Cells werewashed twice and resuspended in PBS containing 0.1% fetal bovine serum and2 @xg/mlpropidium iodide. Cells were analyzed with a FACScan flow cytometer (Becton Dickinson) and a Hewlett-Packard computer using the LYSISprogram.

354

bcl-2 Deficiency in Mice Leads to Pleiotropic Abnormalities: Accelerated Lymphoid

Cell Death in Thymus and Spleen, Polycystic Kidney, Hair Hypopigmentation, andDistorted Small Intestine1

Shinji Kamada, Akihiko Shimono, Yoshitaka Shinto, Tohru Tsujimura, Takeshi Takahashi, Tetsuo Noda,Yukihiko Kitamura, Hisato Kondoh, and Yoshihide Tsujimoto2Department of Medical Genetics, Biomedical Research Center (S. K., I'. S., T. Ta., I'. TI, and Department of Pathology fT. Ts., I'. K.J, Osaka University Medical Schoo4 2-2,Yamadaoka; Institute for Molecular and Cellular Biology. Osaka University, 1-3, Yantadaoka, Suita, Osaka 565 [A. S., H. K.]: and Department of Cell Biology, Cancer institute,Tokyo 103. Japan fT. N.J

ABSTRACT MATERIALS AND METHODS

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bcl-2 DEFICIENCY IN MICE LEAD5 TO PLEIOTROPIC ABNORMALITIES

ATargeting vector

RI

reactions (22) (Fig. 1A). The bcl-2a mRNA is derived from theprocessing of the three exons and produces a protein, Bcl-2a, of 236amino acids. The bcl-2(3 mRNA is derived from the two exons, I andII, producing a protein, Bcl-2(3, of 199 amino acids. Since the targeting vector, pBcl, deleted almost the entire coding exon II, neitherBcl-2a nor Bcl-2f3 protein is produced. After introduction of thetargeting construct (Fig. IA) into E14 ES cells, the resultant C418-resistant clones were initially screened for homologous recombinationby PCR analysis. Disruption of the bcl-2 gene by the targetingconstruct was confirmed by Southern blot analysis. Probes locatedupstream of the 5'-end of the short arm of the targeting constructdetect a 9-kilobase EcoRI fragment from wild-type DNA and a

•‘—lI::J—mutant-specific fragment of 6 kilobases (Fig. 1B). Of about 500C418-resistant ES clones screened, one had the desired disruptedbcl-2 allele. Chimeric animals were generated by injection of heterozygous ES cells into blastocysts recovered from the matings ofB6C3F1 and C57BL16 mice. The contribution of the injected cells tothe germ cells of chimeras was determined by breeding with ICRmice. The heterozygous ES clone produced chimeras which transmitted the bcl-2 mutation to the next generation. Male and female miceheterozygous for the targeted mutation grew and bred normally,

indicating that loss of one functional bcl-2 allele had no overt effects.Fig. 1C shows a representative Southern blot of DNA from offspringof a heterozygous mating. Heterozygous mating yielded 21 homozygous bcl-2@, 42 heterozygous bcl-2@, and 23 wild-type bcl-2@offspring roughly in a Mendelian ratio. Western blot analysis usingpolyclonal antibodies against glutathione S-tmasferase-Bcl-2f3 revealed detectable amounts of Bcl-2 protein in thymus and spleenextracts from bc1-2@ and bcl-2@ animals, but not in those frombcl-2' animals (Fig. 1D), confirming the bcl-2 null mutation. Bcl-2protein levels were higher in bcl-2@@ than in bcl-2@ mice, mdicating a dose-dependent expression of bcl-2 alleles.

Phenotypic Analysis of bcl-2@ Mice. All offspring derivedfrom crosses of heterozygous bcl-2@ mice appeared normal in thefirst week after birth. At about 10 days after birth, some of the animalswere already smaller; these animals were homozygous bcl-2@ mice.Fig. 14 shows the difference in body size between a 17-day-oldbcl-2@ mouse and a bcl-2@@ littermate. About 70% of bcl-2@mice died at 2—3weeks, just before weaning. About 20% of bcl-2@mice survived for 5—7weeks, and the rest (about 10%) lived longerthan 10 weeks. Most bcl-2@ mice had small external ears relative tobody size (Fig. 2B).

Abnormalities in the Thymus and Spleen. Fig. 3 compares thymus and spleen from bcl-2@ mice with those from bcl-2@ mice.Because of accelerated apoptotic cell death of lymphocytes, the sizesofthethymusandspleenfrombcl-2@miceweredramaticallydecreased (Fig. 3A). bcl-2@ thymocytes and splenic lymphocytesdisplayed shorter lives in an in vitro culture system compared withcells from bc1-2@ mice (data not shown). Thymocytes of wastedbcl-2@ mice lost the CD4@/CD8@ double-positive subpopulation(compare Fig. 3B, c with Fig. 3B, d), and splenic lymphocytes lostdifferentiated B220@fIgM4@B cells (compare Fig. 3C, c with Fig. 3C,d). However, the expression of these antigens appeared normal in thespleen and thymus from healthy bcl-2' mice (Fig. 3B, a and b; Fig.3C, a and b). These results suggest that development and maturationofBandTlymphocytescanproceednormallyintheabsenceofBcl-2but that Bcl-2 is indispensable for a normal life span of lymphocytes,at least in the thymus and spleen.

Change in Hair Coat ofbcl-2@ Mice. Because of the genotypesdetermining the coat color of 129/Ola (A'7A―,b/b,@ p/p) andICR (c/c) mice, the coat color of offspring derived from crosses ofheterozygous bcl-2@@ mice was light chinchilla, white, and lightyellow. The coats of bc1-2@ mice that lived longer and initially had

355

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+1+ +1- -I.- +1-. -I-Fig. 1. Targeting the mouse bcl-2 gene. A, schematic diagram of the strategy used to

disrupt the bcl-2 locus. Open boxes, exons of the bcl-2 gene; closed boxes, regionsencoding Bcl-2a protein; hatched boxes, exon specific for the bcl-2@ mRNA; thickhorizontal lines, bcl-2 genomic sequences; crossed lines, putative crossovers between theendogenous bcl-2 locus and the targeting vector pBcl; thick horizontal arrows, thePGK-neo-polyadenylated cassette (Neo) and the PGK-thymidine kinase-polyadenylatedcassette (TK); thin line, bacterial plasmid sequence; small thin line, the region amplifiedby PCR; small thick line, the 1.8-kilobase EcoRI-HindIII DNA probe used for Southernblotanalysis.Thebcl-2aand-@mRNAsareshownunderthewild-typeallele.Cleavagesites for restriction endonuclease EcoRI (RI) are indicated. B, Southern blot analysis of aPCR-positive 0418R@ cellclone. Genomic DNASextracted from a PCR-positive G4l8'@Es cellclone(Lane2) or untransfectedE14EScells(Lane1) weredigestedwithEcoRIand subjected to Southern blot analysis. The wild-type bcl-2 allele generates a 9-kilobasefragment, whereas the targeted allele yields a 6-kilobase fragment. C, genotypes ofprogeny from a bcl-2'@ cross, showing wild-type (Lane 3), heterozygous bcl-2―mutant(Lane2), and homozygousbcl-2@@mutant(Lane1). D, expressionof Bcl-2protein in bcl-2 mutant mice. Spleen (Lanes 1, 2, and 3) and thymus (Lanes 4 and 5) wereisolated from either bcl-2@―(Lane 1), &l-2― (Lanes 2 and 4), or &l-2@' (Lanes 3and5) mice,andtotalproteins(30 @&geach)weresubjectedtoWesternblotanalysisusingpolyclonal anti-glutathione S-transferase-Bcl-2@antibodies.

Histological Analysis. Tissues for light microscopy were fixed in 10%neutral buffered formalin and processed routinely for paraffin embedding.Sections 5 @A.fflthick were stained with hematoxylin and eosin.

Targeting the Mouse bcl-2 Gene and Generation of bcl-2 Mutaut MIce. The mouse bcl-2 gene consists of three exons, whichencode two proteins through different RNA termination and splicing

RESULTS

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light chinchilla hair turned white in the second follicle cycle (Fig. 4).This hypopigmentation began at the chin and proceeded to the hip.Examination of white hair areas under a light microscope indicated alack of melanin in the white hair (data not shown).

Abnormalities of the Kidney. A dramatic consequence of thebcl-2 mutation was observed in the kidneys. Kidneys from symptomatic bcl-2' mice were pale and cystic. Histological analysis revealed many large cysts and dilation of both distal and proximalconvoluted tubules (Fig. 5A), which are typical for polycystic kidneydisease. There were some exfoliated cells inside convoluted tubules,which probably derived from epithelium of the tubules. Only fewrecognizable glomeruli were found (Fig. SB). The kidney architecturewas so distorted that renal failure is probably one of the causes ofdeath in short-lived bcl-2@ mice.

Abnormalities of Intestines Since Bcl-2 is highly expressedinepithelial cells of the small intestine crypt and since its expression isthought to prolong the life span of intestinal progenitor cells (16),abnormalities in the intestine were not unexpected. In control mice,villi ofthe small intestine grew straight to the center of the lumen (Fig.SC). In contrast, villi of bc1-2@ mice were bizarre in shape andapparently shorter than those of control mice (Fig. SD). The intestinallumen of bc1-2@ mice demonstrated clusters of apoptotic cells

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spleen (top) from a 5-week-old bcl-2@ wasted mouse (6.4 g body weight) (left), abcl-2@@ littermate (14.5 g body weight) (right), and a 2-week-old bcl-2'@ mouse (6.9g body weight)(middle). B, flow cytometric analysis ofthymocytes from(a)a 5-week-oldbcl-2@' mouse, (b) a 5-week-old bcl-2@ healthy mouse, (c) a 3-week-old bcl-2@'mouse, and (d) a 3-week-old bcl-2@ wasted mouse (phycoerythrin for CD4; Flit forCD8). C, flow cytometric analysis ofspleniclymphocytes from(a)a 2-week-old bcl-2@'mouse, (b) a 2-week-old healthy bcl-2@ mouse, (c) a 3-week-old bcl-2@@mouse, and(d) a 3-week-old bcl-2@ wasted mouse (FITC for 1gM; phycoerythrin for B220).

(Fig. SD). Apoptosis of epithelial cells occurred only at the villus tipin the control mice, whereas the presence of apoptotic cells was notrestricted to the villus tip in bcl-2@ mice (Fig. SF). It is known that

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bcl-2 DEFICIENCY IN MICE LEAD5 TO PLEIOTROPIC ABNORMALITIES

Bcl-2 is expressed in a wide variety of fetal tissues during embryonic development (17). However, the homozygous bc1-2@ miceappeared to develop normally to birth, and the proportions of wildtype, heterozygous, and homozygous mutant offspring were consistent with Mendelian ratios. Recently, several genes with bcl-2-relatedsequences have been identified (26, 27), and bcl-x, like bcl-2, has beenshown to suppress apoptosis at least upon growth-factor depletion(28). These related genes might compensate for the loss of bcl-2function during embryonic development.

The Bcl-2 protein is expressed in immature CD4/CD8 thymocytes and down-regulated in CD4@/CD8@ thymocytes. Bcl-2 is reexpressed in selected mature single-positive thymocytes (29). Thus, theprotein appears to play a role in lymphocyte development and maturation. However, lymphocyte development initially appeared normalin bcl-2@ mice, suggesting that lymphocytes can differentiate andmature in the absence of Bcl-2, although more detailed study isneeded. The most drastic abnormalities in the lymphoid system werethe dramatic decreases in the sizes of the spleen and thymus, withmassive apoptotic lymphocytes. Lymphocytes from the spleen orthymus of wasted bcl-2@ mice lost the differentiated B220@7IgM@B cell or CD4@/CD8@ T cell subpopulation, respectively. In addition,thymocytes from bc1-2@ mice revealed accelerated cell death invitro (data not shown). These results suggest a causal relationshipbetween the short life span of the lymphocytes and atrophy of thespleen and thymus of bcl-2@ mice. Thus, the loss of Bcl-2 functiondoes not seem to impair development and maturation of lymphocytesbut instead profoundly affects normal life span of lymphocytes.

The bcl-2' mice developed polycystic kidney disease, althoughthe severity and age at onset varied among individual animals. Itremains unknown exactly how the absence of Bcl-2 function leads tothis disease. In the fetal kidney, Bcl-2 has been detected in developingproximal nephrons, glomeruli, and tubular epithelium (17). Recently,it was reported that extensive cell death was involved in the developing kidney (30, 31). Together, these observations suggest an important role for Bcl-2 in kidney development.

A significant consequence of the bcl-2 mutation was the change inhair coat color in the second follicle cycle. Examination of white hairregions of the bcl-2@ mice indicated a lack of melanin in the hair.Melanogenesis in mice is controlled by several gene products, including tyrosinase encoded by the c locus and the product of the b locus,which has been suggested to possess a catalase activity (32). Thehypopigmentation phenomenon has been applied to support a possiblerole of bcl-2 in an antioxidant pathway (19, 33, 34). Our examinationofhairfolliclesafterthesecondfolliclecycleshowedacompletelackofmelanocytesinhairfolliclesofthebc1-2@mice,comparedwiththe abundant dopa-positive melanocytes in the control mice.4 Thus,hair hypopigmentation caused by the bcl-2 deficiency appears toresult from the accelerated death of melanocytes and not from impaired melanogenesis by increased oxidant species.

Villi ofthe small intestine were apparently shorter in bcl-2@ micethan in normal control mice. Moreover, the villi of bc1-2@ micewere bizarre in shape, and epithelial cells were not well aligned. Theepithelial layer of the intestine consists of several different types ofcells, all of which derive from progenitor cells at the crypt. Daughtercells of progenitors divide rapidly and differentiate as they moveeither upward or downward. The shortness and bizarre shape of thevilli may be attributable to the decrease of dividing cells in the cryptsand exfoliation of epithelial cells, which was not restricted to thevillus tip. Because a high level of Bcl-2 expression has been reportedin the crypts (16), the decrease of dividing cells in the crypts and the

4 Data in preparation.

Fig. 4. Change in coat color of bcl-2@ mice. A, a bcl-2@ mouse at 41)days. B, thesame mouse at 50 days.

there are only a small number of progenitor cells with proliferativecapacity at crypts, from which all cells at epithelial layers are derived.The number of mitoses in crypts was analyzed for bcl-2' andcontrol mice. As shown in Table 1 and Fig. 5, E and F, the number ofmitoses was significantly smaller in the crypts of bc1-2@@ mice thanin those of normal control mice, suggesting that epithelial progenitorcells at crypts were lost in the absence of Bcl-2 protein. The numberof mitoses in crypts of small intestine varied considerably among thebcl-2@ mice; but as described in the previous sections, a variation inseverity of abnormalities among bcl-2@ mice was common in othertissues. In both control and bcl-2@ mice, both absorptive epithelialcells and goblet cells were found in villi, but the proportion of gobletcells appeared to be larger in bc1-2@ mice than in control mice.Paneth cells were found in the bottom of crypts in both bc1-2@ miceand control mice (Fig. 5, E and F).

DISCUSSION

The lines of bcl-2-deficient mice have been described (19, 20). Wehave independently generated mice lacking Bcl-2a and -f3 proteins byspecifically targeting the bcl-2 sequences encoding almost the entiresecond coding exon. Western blot analysis indicated the absence ofdetectable Bcl-2 in homozygous bc1-2@ mice, and heterozygousbcl-2@' mice displayed reduced levels (about 50%) of Bcl-2 ascompared with wild-type mice. The heterozygous bcl-2@' miceappear to be completely normal after 6 months of age.

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bc!-2 DEFICIENCY IN MICE LEADS TO PLEIOTROPIC ABNORMALITIES

Fig. 5. Histological features of the kidney andsmall intestine of a bcl-2@ mouse and a normallittermate control. Stained with hematoxylin andeosin. A, polycystic kidney of a 5-week-old bcl2@ mouse. Original magnification, X 18. B,high-power view of the polycystic kidney. Thenumber of glomeruli is remarkably reduced(arrow),andproximalconvolutedtubulesaredilated.Arrowheads,exfoliatedapoptoticcells.Original magnification, x 90. C, small intestine of the5-week-old bcl-2@' mouse, showing normallength and shape of villi. Original magnification, X 185. D, small intestine of the 5-week-oldbcl-2@ mouse. Viii are short and bizarre inshape. Star, a cluster of apoptotic epithelial cells.Original magnification, X 185.E, high-power viewof the small intestine of the control mouse. Severalmitoses are found in the crypts (arrows). Originalmagnification, x 490. F, high-power view of thesmall intestine of the &l-2@ mouse. Mitoseswere rarely found in crypts. Exfoliation of apoptotic epithelial cells (arrowheads) does not appearto be restricted to the tip of viii. Original magnification, X 490.

- .

abnormal exfoliation pattern may result directly from the lack of Bcl-2protein. Despite the abnormal architecture of villi and crypts, absorptive epitheial, goblet, and Paneth cells were detectable in the smallintestine of bc1-2@ mice, suggesting that Bcl-2 protein may not beessential for differentiation of the progenitor cells. Only a smallnumber of crypt progenitor cells with proliferative ability support therapid epithelial cell turnover of small intestines. Fewer mitotic figuresseen in small intestines of the bcl-2@ mice than in the control miceare likely the result of accelerated cell death of the progenitors in the

absence of bcl-2, suggesting an essential role of bcl-2 in maintenanceof the long-lived crypt progenitors.

The bcl-2@ mice have a variable, crossbred genetic background.Variations in genetic background may help determine the severity ofthe phenotype, the life span, the body weight, the degree of polycystickidney disease, and the losses of spleen and thymus.

The phenotype of the bcl-2@ mice provides new informationabout the role of the Bcl-2 protein during development and homeostasis. The fact that these independently developed lines of bd-2@

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No. ofmitoses (A)No.

ofcrypts (B)Freque A/Bncy

of mitoses

Av. ±SEbcl-2@166810130.6626329040.70381314420.560.58

±o.osa468517070.4055038640.58bcl-2@62026590.317848950.09811912680.09o.18±0.04a91568680.18101778190.22ap<001

bcl-2 DEFICIENCY IN MicE LEADS TO PLEIOTROPIC ABNORMALITIES

Table 1 Frequencyofmitoses in cryptsof the small intestineCross-sections of the small intestine were cut and stained with hematoxylin and eosin.

Only crypts including the lower halves were counted because the mitoses were restrictedto the lower halves.

12. Monaghan,P., Robertson,D., Amos,T. A. S., Dyer,M. J. S., Mason,D. Y., andGreaves, M. F. Ultrastructural localization ofbcl-2 protein. i. Histochem. Cytochem.,40: 1819—1825,1992.

13. Jacobson,M. D., Burnett,J. F., King,M. D., Miyashita,T., Reed,J. C., and Raff,M. C. Bcl-2 blocks apoptosis in cells lacking mitochondrialDNA. Nature(Land.),361: 365—369,1993.

14. Akao, Y., Otsuki, Y., Kataoka, S., Ito Y., and Tsujimoto, Y. Multiple subcellularlocalization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulummembrane, and mitochondrial membranes@Cancer Rca., 54: 2468-2471, 1994.

15. Krajewski, S., Tanaka, S., Takayama, S., Schibler, M. J., Fenton, W., and Reed, J. C.Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in thenuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res., 53: 4701—4714,1993.

16. Hockenbery,D. M., Zutter,M., Hickey, W., Nahm, M., and Korsmeyer,S. J. Bcl-2protein is topographically restricted in tissues characterized by apoptotic cell death.Proc. Natl. Aced. Sci. USA, 88: 6961-6965, 1991.

17. LeBrun, D. P., Warnke, R. A., and Cleary, M. L Expression of bd-2 in fetal tissuessuggests role in morphogenesis. Am. J. Pathol., 142: 743-753, 1993.

18. Mansour,S.,Thomas,K. R.,andCapecchi,M.R. Disruptionof theproto-oncogeneint-2 in mouse embryo-derived stem cells: a general strategy for targeting mutationsto non-selectable genes. Nature (Land.), 336: 348—352,1988.

19. Veis, D. J., Sorenson,C. M., Shutter,J. R., andKorsmeyer,S. 3. Bcl-2-deflcientmicedemonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmentedhair. Cell, 75: 229—240,1993.

20. Nakayama, K., Nakayama, K-L, Negishi, I., Kuida, IC, Sawa, H., and Loh, D. Y.Targeted disruption of Bcl-2a@ in mice: occurrence of gray hair, polycystic kidneydisease, and lymphocytopenia. Proc. NatL Aced. Sci. USA, 91: 3700-3704, 1994.

21. Doetschman, T., Gregg, R. 0, Maeda, N., Hooper, M. L, Mellon, D. W., Thompson,S., andSmithies,0. Targetedcorrectionof a mutantHPRTgerIe mouseembryonicstem cells. Nature (Land.), 330: 576—578,1987.

22. Negrini, M., Silini, E., Kozak, C., Tsujimoto, Y., and Croce, C. M. Molecular analysisof mbcl-2: structureand expression of the murinegene homologous to the humangene involved in fofficular lymphoma. Cell, 49: 455—463,1987.

23. Rudnicki, M. A., Braun, T., Hinuma, S., and iaenisch, R. Inactivation of MyoD inmice leads to up-regulation of the myogenic HLH gene My/5 and results in apparently normal muscle development. Cell, 71: 383—390,1992.

24. Handyside, A. H., O'Neill, 0. T., Jones, M., and Hooper, M. L Use of BRLconditionedmediumin combinationwith feederlayersto isolate a diploidembryonalstem cell line. Roux's Arch. Dcv. Bini, 198.-48—55,1989.

25. Sawai, S., Shimono, A., Hanaoka, K., and Kondoh, H. Embryonic lethality resultingfrom disruption of both N-myc alleles in mouse zygotes. New BioL, 3: 861-869,1991.

26. Kozopas, K. M., Yang, T., Buchan, H. L, Thou, P.. and Craig, R. W. MCL-l, a geneexpressed in programmedmyeloid cell differentiation,has sequence similarity tobcl-2. Proc. Nati. Acad. Sci. USA, 90: 3516—3520, 1993.

27. Oltvai, Z. N., Milhiman,C. L, and Korsmeyer, S. J. Bcl-2 heterodimerizes in vivowith a conserved homologue, Bax, that accelerates programmed cell death. Cell, 74:609—619,1993.

28. Boise, L H., Gonzalez-Garcia, M., Postema, C. E., Ding, L, Lindsten, T., Turka,L A., Mao, X., Nunez, G., and Thompson, C. B. bcl-x, a bcl-2-relatedgene thatfunctions as a dominant regulator of apoptotic cell death. Cell, 74: 597—608,1993.

29. Veis, D. J., Scntman, C. L, Bach, E. A., and Korsmeyer, S. J. Expression of the Bcl-2protein in murine and human thymocytes and in peripheral T lymphocytes. I.hnmunol., 151: 2546—2554,1993.

30. Koseki, C., Herzlinger, D., and Al-Awqati, 0. Apoptosis in metanephric development.J. Cell BioL, 119: 1327—1333,1992.

31. Coles, H. S. R., Burne, J. F., and Raff, M. C. Large-scale normal cell death indeveloping rat kidney and its reduction by epidermal growth factor. Development,118: 777—784,1993.

32. Halaban, R., and Moellmann, 0. Murine and human b locus pigmentation genesencode a glycoprotein (gp75) with catalase activity. Proc. Natl. Acad. Sci. USA, 87:4809—4813, 1990.

33. Hockenbery, D. M., Oltval, 7. N., Yin, X., Milliman, C. L, and Korsmeyer, S. J. bcl-2functions in an antioxidant pathway to prevent apoptosis. Cell, 75:241-251,1993.

34. Kane, D. J., Sarafian, T. A., Anton, R., Hahn, H., Gralla, E. B., Valentine, J. S., Ord,T., and Bredesen, D. E. Bcl-2 inhibitionof neuraldeath:decreasedgenerationofreactive oxygen species. Science (Washington DC), 262: 1274—1277,1993.

359

mice revealed similar phenotypes supports the conclusion that theabnormalities observed are the consequence of bcl-2 gene disruption.

REFERENCES

1. Tsujimoto, Y., Gorham,J., Cossman, J., Jaffe, E., and Croce, C. M. The t(14;18)chromosome translocations involved in B-cell neoplasms result from mistakes in VDJjoining. Science (Washington DC), 229: 1390—1393,1985.

2. Bakhshi, A., Jensen, J. P., Goldman, P., Wright, J. J., McBride, 0. W., Epstein, A. L,and Korsmeyer, S. J. Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on18. Cell, 41: 899—906,1985.

3. Cleary, M. L, and Sklar, J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint cluster region near atranscriptionallyactive locus on chromosome 18. Proc. Natl. Acad. Sci. USA, 82:7439—7443,1985.

4. Vaux, D. L, Cory, S., and Adams, J. M. bcl-2 gene promoteshaematopoieticcellswvival and cooperates with c-myc to immortalize pre-B cells. Nature (Lend.), 335:440-442, 1988.

5. Tsujimoto, Y. Stress-resistance conferred by high level of bcl-2a protein in human Blymphoblastoid cell. Oncogene, 4: 1331—1336,1989.

6. Nunez, 0., London, L, Hockenbery, D. M., Alexander, M., McKearn, J. P.,and Korsmeyer, S. J. Deregulated bcl-2 gene expression selectively prolongs survivalof growth factor deprived hematopoieticcell lines. J. Immunol., 144: 3602-3610,1990.

7. Hockenbery, D. M., Nunez, G., Milliman, C., Schreibcr, R. D., and Korsmeyer, S. J.Bcl-2 is an inner nsitochondrial membrane protein that blocks programmed cell death.Nature (Land.), 348: 334—336,1990.

8. Sentman,C. L, Shutter,J. R., Hockenbery,D. M., Kanagawa,0., and Korsmeyer,S. J. bcl-2 inhibitsmultipleformsof apoptosisbut not negative selection in thymocytes. Cell, 67: 879—888,1991.

9. Garcia,I., Martinou,I., Tsujimoto,Y., andMartinou,i-C. Preventionof programmedcell death ofsympathetic neurons by the proto-oncogene bcl-2. Science (WashingtonDC), 258: 302—304,1992.

10. Allsopp, T. E., Wyatt, S., Paterson, H. F., and Davies, A. M. The proto-oncogenebcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis.Cell, 73: 295—307,1993.

11. Chen-Levy, Z., Nourse, I., and Cleary, M. L The bcl-2 candidateproto-oncogeneproduct is a 24-kilodalton integral-membrane protein highly expressed in lymphoidcell lines and lyniphomascarryingthe t(14;18) translocation.Mol. Cell. Biol., 9:701—710,1989.

on March 21, 2020. © 1995 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1995;55:354-359. Cancer Res   Shinji Kamada, Akihiko Shimono, Yoshitaka Shinto, et al.   IntestinePolycystic Kidney, Hair Hypopigmentation, and Distorted SmallAccelerated Lymphoid Cell Death in Thymus and Spleen,

Deficiency in Mice Leads to Pleiotropic Abnormalities:bcl-2

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