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A PCR-based Method for the Analysis of Human CD44 Splice Products

D. Henk J. van Weering, Pieter D. Baas, and Johannes L. Bos

Laboratory for Physiological Chemistry, Utrecht University, 3521 GG Utrecht, The Netherlands

CD44 is a transmembrane glycopro- CD44 is a with a molec- vantage of these techniques is that they tein involved in the Interaction be- ular mass ranging from 85 to 200 kD. only show the presence of certain vari- tween cells and . The human CD44 consists of at ant , but they do not reveal in Several variant forms of CD44 exist, least 19 exons, of which at least 12 can which context these exons are present in which differ from each other in the be alternatively spliced. (1). Nine of these each CD44 molecule. It is very possible composition of both the intra- and alternatively spliced or variant exons en- that a CD44 molecule that contains, for extracellular domain of the . code protein segments that can be in- instance, solely the variant v6-en- Post-translational modification and serted as a cassette into the extracellular coded sequences may differ functionally alternative RNA processing are re- domain of the molecule (Fig 1). CD44 is from a CD44 molecule containing se- sponsible for this variation. Recently, the principal surface for hy- quences encoded by exons v6--vl0. With it was found that certain variant aluronic acid, an extracellular matrix the method described in this paper it is CD44 , containing extra se- component. (2) Insertion of three of the relatively simple to analyze the compo- quences in the extracellular domain nine variant exons (v8-vl0) in the extra- sition of each major CD44 mRNA species of the protein, are involved in meta- cellular domain of the CD44 molecule present in a cell type. The technique is static spread of tumor cells. Variant abrogates binding. (3) based on reverse transcriptase-poly- CD44 proteins are also involved in CD44 is present on many different cell merase chain reaction (RT-PCR). We de- immunological functions of T and B types in the body (4,5) and is also in- scribe the exon composition of the ma- cells. A large variety of alternatively volved in trafficking (for re- jor CD44 mRNAs expressed by six spliced CD44 mRNAs can be ex- view, see ref. 6). In addition, it was different human cell lines. pressed by cells. We have developed a shown that CD44 variants containing method for the analysis of CD44 the protein domain encoded by exon v6 MATERIALS AND METHODS mRNAs present in the cell. This re- are transiently expressed during the ac- verse -polymerase chain tivation of B cells, T cells, and macro- Tumor Cell Lines reaction (RT-PCR)-based method can phages. against the exon v6 In this study six different human cell be used to analyze the exon composi- domain of CD44 can interfere with the lines were used: HT29 (colon carci- tion of each major CD44 mRNA species immunological function of cells express- noma), HT29D4 (colon carcinoma), (]4) present in the cell. In this study we de- ing these CD44 molecules. (7) It was also SW613 (colon carcinoma; obtained from scribe the analysis of CD44 mRNAs iso- found that expression of isoforms of P.E. Holthuizen, Laboratory for Physio- lated from six different human cell CD44 that contain sequences encoded logical Chemistry, Utrecht, The Nether- lines. by the variant exons v6-v7 can confer lands), Hep3B (hepatocellular carcinoma; metastatic potential to nonmetastatic rat also obtained from P.E. Holthuizen), tumor cells, (8-1~ indicating that aber- HaCaT (spontaneously immortalized ke- rant expression of variant CD44 may ratinocytes), and HeLa (cervix carci- play a role in tumor . These noma). Cells were grown in Dulbecco's results show the relevance of alternative modified Eagle medium (DMEM) sup- splicing of CD44 in normal cell func- plemented with 10% fetal calf serum tion, as well as in tumor progression. (FCS). Extra glucose (4.5 g/l) was added In several studies human tumors have for the HT29D4 cell line. been screened for the expression of al- ternatively spliced forms of CD44. (s'11-13) RNA Isolation Variant exons were detected by blot hy- bridization with variant exon-specific Total cellular RNA was isolated using probes or by staining of CD44 proteins RNA-SR (Biogenesis, Bournemouth, En- containing variant exon-encoded se- gland) according to the manufacturer's quences with antibodies. A major disad- protocol. Briefly, -107 cells were col-

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C 13 pv2 pv3 pv4 pv5 pv6 pv7 pv8 pv9 pv 10 C2A C 12A --~ >.~ ~ ---> .--~ ~ ---~ ---~ --~ ~__ _ ~--.- cause the RNase H treatment did not in- ! I1[ fluence the PCR amplification effi- ciency, we did not include this step in a i the standard protocol (data not shown). s I 5 6 7 8 9 10 11 12 13 14 i 15 16 17 (171/ (129) (126/ (114) (117) (129) (132) (102) (90) (204) (63) (72) (18/ 79) 102) 58) PCR Amplification Primers for PCR amplification were syn- v2 v3 v4 v5 v6 v7 v8 v9 vlO thesized using the phosphoramidite method on a Gene Assembler (Pharma- I I I I I I I I I cia). Primers were chosen at the 5' side of 1 II ill IV V each variant exon, except for exon v3, I I where we chose one primer upstream const, variant exons ~ constant exons exons (pv3 I) and one primer downstream of the alternative acceptor site (pv3 H) (Ta- FIGURE 1 Schematic representation of a part of the human CD44 gene. From top to bottom are ble 1). shown primers (arrows), the exons (solid bars) numbered according to Screaton et al., (1) and the size of the individual exons. For constant exon 5, the size is given starting from primer C13, PCR reactions were performed under including or excluding the 92 nucleotides after the alternative donor splice site (d). For Constant mineral oil in a total volume of 100 i~l exon 17, the size is given until the end of primer C2A/C12A, respectively. In exon 7 (v3), an containing 50 mM KC1, 20 mM Tris-HC1 alternative acceptor site is present, and the size of this exon is given including or excluding the (pH 8.3), 2.0 mM MgClz, 50 ~I,M of each first 24 nucleotides. Below the exon sizes, the variant exon nomenclature as suggested by T61g et dNTP (Pharmacia), 20 pmoles of each al., (9) and the domain numbers as reported by Hofmann et al. (16) are shown. primer, 2 units of Taq polymerase (Per- kin-Elmer Cetus), and 1 i~l of cDNA (the equivalent of 50 ng RNA). Primers were added when the reaction mixture was at lected by trypsinization, and RNA was facturer's protocol. Briefly, 1 I~g of total a temperature of 95~ ("hot start"). The isolated using RNA-SR, a guanidinium RNA was mixed with DEPC-treated H20 , cDNA was amplified in 30 cycles; 0.5 thiocyanate- and phenol-containing re- a CD44-specific oligonucleotide at the 3' min denaturation at 95~ 1.5 min an- agent. On average, 100 I~g of total RNA side of constant exon 17 (C12A) (20 nealing at 56~ and 1 min extension at was isolated from 107 cells using this iso- pmoles), and 20 units of RNasin (Boeh- 73~ followed by a final extension at lation protocol. ringer). The mixture was incubated at 73~ for 10 min using a Bioexcellence 70~ for 5 min; subsequently, dNTPs (fi- DNA incubator. In the experiment nal concentration 1 mM), RT buffer, DTT shown in Figure 5, below, the cDNA was (5 mM), and 200 units of Superscript Plus amplified in 35 cycles. PCR products For cDNA synthesis several different RTs were added to make a total volume of 20 were analyzed on a 1.25% agarose gel were tested: avian myeloblastosis virus l~l. The mixture was incubated at 37~ (Sigma) in 1• TBE. In the negative con- RT (AMV-RT), Moloney murine leuke- for 1 hr, followed by inactivation of the trol no cDNA was added to the PCR re- mia virus RT (Mo-MLV-RT), and an enzyme at 95~ for 5 min. In some ex- action. RNase H- Mo-MLV-RT. Best results were periments the mRNA template was de- obtained with the RNase H- Mo-MLV- graded by incubation with 60 units of RT Superscript Plus (GIBCO-BRL). cDNA RNase H (Amersham) for 20 min at 37~ was synthesized according to the manu- before enzyme inactivation at 95~ Be- Exon-specific Runoff Analysis For the exon-specific runoff analysis of the PCR products, 1 i~l of the C13-C2A TABLE 1 Oligonucleotides Used for cDNA Synthesis and PCR Reactions PCR product was reamplified using lin- ear PCR. The linear PCR reaction was Oligonucleotide Sequence (5' ~ 3') performed in a total volume of 10 i~l C2A CCA AGA TGA TCA GCC ATT CTG G containing 50 mM KCI, 20 mM Tris-HC1 C12A ATG CAA ACT GCA AGA ATC (pH 8.3), 2.0 mM MgClz, 10 I~M of each C13 AAG ACA TCT ACC CCA GCA AC dNTP, 0.2 pmole of a TLC-purified, 3Zp_ pv2 GAT GAG CAC TAG TGC TAC AG labeled exon-specific primer, and 0.5 pv31 ACG TCT TCA AAT ACC ATC TC unit of Taq polymerase. The DNA was pv3 II T GG GAG CCA AAT GAA GAA AA amplified in 20 cycles of 0.5 min at 95~ pv4 T CA ACC ACA CCA CGG GCT TT 0.5 min at 56~ 1 min at 73~ followed pv5 GTA GAC AGA AAT GGC ACC AC by 10 cycles of 0.5 min at 95~ 1.0 min pv6 CAG GCA ACT CCT AGT AGT AC at 73~ and a final extension at 73~ for pv7 CAG CCT CAG CTC ATA CCA GC 10 min. The 3zP-labeled runoff products pv8 TCC AGT CAT AGT ACA ACG CT pv9 CAG AGC TTC TCT ACA TCA CA were analyzed on a 7 M urea-3% poly- pvl0 GGT GGA AGA A GA GA C C CA AA acrylamide gel in a Bio-Rad Sequi-gen se- quencing gel system.

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Cycle Sequencing of PCR Products in combination with one or more up- or listed all of the possible exon combina- downstream variant exons. In addition, tions and their sizes. This list is available For sequence analysis of PCR products, variant exon v3 contains an alternative on request. DNA bands were cut from a 1.25% low splice acceptor site, and in the 5' con- To analyze the composition of each melting point agarose gel (Sigma). The stant part of the gene (exon 5) an alter- amplified band (representing one mRNA cycle sequencing reaction was per- native splice donor site is present. Vari- species), two different amplification formed in a total volume of 10 ~l con- ant exon v3 is divided into v31 and v3 ~. methods can be used. In the first method taining 50 mM KC1, 20 mM Tris-HC1 (pH The constant exon 5 is called C (constant (exon-specific runoff analysis), the PCR 8.3), 2.0 mM MgC12, 10 ~M of each dNTP, sequence) or C' when the alternative product from the amplification between 0.2 pmole of TLC-purified, 3ZP-labeled splice donor site is used. When no vari- primers C13 and C2A is reamplified by sequence primer (C13), 0.5 unit of Taq ant exons are included in the CD44 linear PCR using ~ZP-labeled oligonucle- polymerase, and 300 ~M ddATP or 200 mRNA, this form is called the standard otides pv2-pvl0 plus C13. This amplifi- ~M ddCTP, or 100 ~M ddGTP, or 400 FLM CD44 or CD44s. cation yields runoff products from the ddTTP (Boehringer), and 6 ~l of the DNA In the method we have developed, variant exon-specific primer to the end containing gel slice. The DNA was am- cDNA synthesized with primer C12A is of the PCR product. The runoff products plified in 20 cycles of 0.5 min at 95~ 0.5 amplified using two primers located out- of all the exon-specific primers are ana- min at 56~ 1 rain at 73~ followed by side the variant part of the CD44 mRNA lyzed on polyacrylamide gels to deter- 10 cycles of 0.5 rain at 95~ 1.0 min at (C13 and C2A) (Fig. 1). This amplifica- mine their exact length. In the second 73~ and a final extension at 73~ for 10 tion yields several different products de- method (exon-specific PCR analysis), the min. The sequence products were ana- pending on the cell type analyzed. Some cDNA is amplified by exponential PCR lyzed on a 7 M urea-5% polyacrylamide cell types express a single CD44 mRNA between primer C2A and each of the gel in a Bio-Rad Sequi-gen sequencing species, and others express various CD44 variant exon-specific primers pv2-pvlO. gel system. mRNAs, resulting in several different These amplification products can be an- amplification products (Fig 2A). Calcula- alyzed directly by agarose gel electro- tion of the molecular sizes of these am- phoresis. Therefore, in both methods RESULTS AND DISCUSSION plification products does not provide the cDNA is amplified between one Method sufficient information to determine the primer in the constant part of the mRNA exact exon composition of the bands, be- and one primer in each variant exon. De- In Figure 1, a schematic representation is cause, theoretically, 768 possible combi- termination of the length of the exon- given of exons 5-17 of the human CD44 nations of exons can be formed with the specific amplification products from the gene. (1) Different nomenclatures for the variant exon v2-vlO (including the alter- agarose or polyacrylamide gel enables variant exons are in use. In this report native acceptor site in variant exon v3). the determination of the composition of the nomenclature suggested by T61g et Many of these 768 combinations have the amplified bands. A combination of al. is used. (9) Via , approximately the same size. However, the results of all the variant exon-specific each of the variant exons v2-vlO can be in practice we observe only a small sub- primers reveals the composition of the inserted into the mRNA, either alone or set of these exon combinations. We have C13-C2A bands and, thus, of the mRNAs present in the cell. Therefore, three pieces of information are combined in -4 B the determination of the composition of X Y 10 9 8 7 6 5 4 3 2 C13 each band: (1) the first exon of the am- r,-1 ======,= =:= plified product, (2) the size of the entire m product, and (3) the presence of a corre- sponding amplification product from i m C-5-C one of the other (more 3') primers. Be- m I cause RT PCR is used in this method, it cannot be used for quantification of in- dividual mRNA species in a cell; only the composition of the mRNAs can be deter- mined. 10 9 8 7 6 5 4 3 2 C13 In Figure 2, an example of a hypothet- ical RNA analysis is given. RNA X con- C-2-3-4-5-6- D u 7-8-9-10-C tains one C13-C2A band, so in these m cells only one CD44 mRNA species is m m C-3-4-8-10-C C-8-9-10-C present (Fig. 2A). Amplification with all of the variant exon-specific primers, ei- C-6-C ther by exon-specific runoff or by exon- FIGURE 2 Schematic representation of an RNA analysis. (A) X and Y represent two different specific PCR, shows that only exon v5 is hypothetical RNA samples amplified between primers C13 and C2A. (B) Exon-specific runoff present in the cDNA (mRNA) (Fig. 2B). analysis of RNA X, showing the presence of only variant exon v5 in the mRNA. (C) Exon-specific The DNA fragment amplified between runoff analysis of RNA Y, showing the composition of the four mRNAs amplified between primers C13 and C2A thus consists of 5'- primers C13 and C2A (see text for explanation). constant part-variant exon v5-3'-con-

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yield of amplification product of the HT29D4 cell line is lower. Both cell lines express eight major products of which the band running at -700 bp has the highest intensity. A band of similar length is also seen after amplification of mRNA from the HaCaT cell line (band D). The cervix carcinoma cell line HeLa and the colon carcinoma cell line SW613 FIGURE 3 Ethidium bromide staining of the C13-C2A amplified products of the cell lines ana- express only one major product with an lyzed in this study. (Right) The molecular mass marker (k-DNA x EcoRI-HindlII + pAT153 x electrophoretic mobility of -325 bp. Hinfl) is shown. (A,C,D,E at left) The major amplification products of the HaCaT cell line. These These cell lines show other CD44 mRNA letters correspond to the products mentioned in Table 2 and Fig. 4. species only very faintly. RNA isolated from the hepatocellular carcinoma cell line Hep3B does not show any amplifi- stant part (C-v5-C). RNA Y shows four runs at a length of -1350 bp. The colon cation product. This was not surprising different bands after amplification with carcinoma cell lines HT29 and its sub- because normal hepatocytes do not primers C13 and C2A (Fig. 2A). Amplifi- clone HT29D4 show nearly identical express detectable amounts of CD44 cation with the variant exon-specific CD44 mRNA expression; only the total proteins. (4,s) cDNA synthesis and ampli- primers (Fig. 2C) shows that exon vl0 is present (vl0-C). Exon v9 is present only in combination with exon vl0 (v9-vl0- C), which can be concluded from the size of the product and the presence of the vl0-C product. Exon v8 is present in combination with exons v9 and vl0 (v8- v9-vl0-C), but also in combination with only exon vlO (v8-vl0-C), again con- cluded from the size of the bands and the presence of the two products one exon shorter on the 3' side (v9~ and vl0-C). Exon v6 is present in com- bination with exons v7, v8, v9, and vl0 (v6-v7-v8-v9-vl0-C) but also without any of the other alternatively spliced exons (v6-C). In this way, for each exon it is determined in which context it is pres- ent in the mRNA. Then, by combining the data of the size of the C13-C2A bands with the data for each exon it is possible to determine the exact exon composition of the major mRNA prod- ucts expressed by a certain cell type.

Analysis of CD44 mRNA Present in Cell Lines CD44 mRNA expressed by six different human cell lines was analyzed using the method described above. The following cell lines were tested: HaCaT, HT29, HT29D4, HeLa, SW613, and Hep3B. To- tal RNA was isolated from these cell lines, followed by cDNA synthesis, cDNA was amplified between primer C13 and C2A, giving amplification products of each CD44 mRNA species present in the cells (Fig. 3). The spontaneously immor- FIGURE 4. Exon-specific runoff analysis of the HaCaT (A) and HT29D4 (B) cell lines using 32p. talized keratinocytes (HaCaT cell line) labeled oligonucleotides. (Top) The primers are shown. Next to the molecular mass marker showed four major bands (A, C, D, E). (k-DNA x AvaII), the letters A-J indicate the major amplification products listed in Table 2 (A-E) The top band with the highest intensity and Table 3 (F-J).

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fication with TATA-binding protein From these data it is clear that differ- exons are present in each mRNA species, (TBP)~ primers were used as a ent CD44 splice products are expressed we used the exon-specific runoff analysis positive control for the quality of the in different cell lines. However, from the described above. RNA preparations. From the Hep3B cell length of the products alone it is not pos- line, TBP mRNA could be amplified, sible to determine what the exon com- Exon-speciflc Runoff Analysis showing that the RNA preparation was position is of the individual amplifica- intact (data not shown). tion products. To determine which In Figure 4, the result of an exon-specific runoff analysis is shown. HT29D4 and HaCaT cDNAs, amplified between prim- TABLE 2 Analysis of mRNAs in the HaCaT Cell Line by Exon-specific ers C13 and C2A, were used as a template Runoff Analysis for linear PCR with SzP-labeled variant exon-specific primers pv2-pvlO. Radio- Exon Measured Calculated active amplification products were ana- primer size Composition size lyzed by polyacrylamide gel electro- pvlO 336 vlO-C 340 phoresis. By determining the size of the pv9 432 v9-vlO-C 433 major bands of each amplification and 226 v9-C 229 by combining these data with that from pv8 538 v8-v9-vlO-C 541 the more 3' variant exon-specific ampli- 451 v8-vl0-C a 451 fication products, the composition of 334 v8-v9-C 337 the major mRNAs in these cells could be 245 v8-C 247 pv7 691 v7-v8-v9-vlO-C 681 deduced. In Table 2, the complete anal- 591 v7-v8-vlO-C 591 ysis of CD44 mRNA expressed by the 476 v7-v8-v9-C 477 HaCaT cell line is shown. For each 385 v7-v8-C 387 primer, the measured size of the ampli- pv6 821 v6-v7-v8-v9-vl0-C 808 fied products is shown. The most likely 601 v6-v7-v8-v9-C 604 combination of variant exons that 512 v6-v7-v8-C 514 match the size of the amplified product pv5 907 v5-v6-v7-v8-v9-vlO-C 925 was deduced by combining the follow- 793 v5-v7-v8-v9-v 10-C 796 ing three pieces of information: (1) the 721 v5-v6-v7-v8-v9-C 721 size of the amplified product, (2) the first 622 v5-v6-v7-v8-C 631 pv4 1033 v4-v5-v6-v7-v8-v9-v 10-C 1039 exon (determined by the primer), and 912 v4-v5-v7-v8-v9-vlO-C 910 (3) data from amplifications with more 834 v4-v5-v6-v7-v8-v9-C 835 3' primers. In this way, the composition 747 v4-v5-v6-v7-v8-C 745 of the bands amplified with primer C13, pv3 n 1136 v3U-v4-v5-v6-v7-v8-v9-vl0-C 1138 representing the mRNA species present 1016 v3U-v4-v5-v7-v8-v9-v10-C 1009 in the cell lines, was deduced. 924 v3n-v4-v5-v6-v7-v8-v9-C 934 In contrast to the results obtained by 843 v3n-v4-v5-v6-v7-v8-C 844 Hofmann et al., (16) we found that the 782 v3U-v7-v8-v9-vlO-C 778 major CD44 mRNA product in the 644 v3n-v8-v9-vlO-C 646 HaCaT cell line is CD44v3I-v10 (product 245 v31I-C 250 pv3 ~ 1166 v31-v311-v4-v5-v6-v7-v8-v9-vlO-C 1165 A), including the first 24 nucleotides 1048 v31-v3~I-v4-v5-v7-v8-v9-vlO-C 1036 from the 5' part of exon v3 (v3I). We 952 v3l-v3II-v4-v5-v6-v7-v8-v9-C 961 found no evidence for the presence of 867 v31-v3U-v4-v5-v6-v7-v8-C 871 mRNA species lacking these 24 nucle- 808 v3I-v3n-v7-v8-v9-v 10-C 805 otides, either in the exon-specific runoff 675 v3I-v3n-v8-v9-vlO-C 673 analysis, or after sequencing of the PCR 281 v3I-v3n-c 277 products. Also, in normal keratinocytes, pv2 1281 v2-v31-v3II-v4-v5-v6-v7-v8-v9-vlO-C 1292 the exon v31 part is present in the C13 1458 C-v2-v3I-v311-v4-v5-v6-v7-v8-v9-vl0-C 1467 mRNA. (17) In Table 3, the major mRNA 1331 C-v3~-v3n-v4-v5-v6-v7-v8-v9-v 10-C 1338 (A)b species expressed by the other cell lines 1216 C-v4-v5-v6-v7-v8-v9-vlO-C 1212 (B)b 980 C-v6-vT-v8-v9-vlO-C 981 (C) b are listed (only the C13-C2A products 853 C-v7-v8-v9-vl0-C c 852 are shown). The results shown in Table 3 C-v3~-v3n-v8-v9-vl0-Cc 846 were also obtained using the exon-spe- 724 C-v8-v9-vlO-C 720 (D) b cific runoff analysis. For the HT29 and 524 C-vlO-C 528 HT29D4 cell lines, the most abundant 512 C-v8-v9-C 516 product is CD44v8-v10 (product H). 320 C-C 324 (E)b This is in agreement with several other reports. (x's's'16,as) We did not observe aBecause of the low intensity of the band on the original film it is not visible on the reproduction. bThe letters A-E refer to the same letters in Figs. 3 and 4. qualitative differences in CD44 expres- cUsing this technique it was not possible to determine which of these two exon combinations is sion between these two cell lines, only a represented by this band. difference in the total yield of amplified

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TABLE 3 Results of the Analysis of mRNA Species in the HT29, HT29D4, HeLa, and exon 5. In the HT29, HT29D4, HeLa, and SW613 Cell Lines by Exon-specific Runoff Analysis SW613 cell lines only a very low expres- sion of C'-C mRNA was observed, show- Cell Measured Calculated ing the infrequent use of the alternative line size Composition size splice donor site in constant exon 5. HT29/HT29D4 1327 C-v3Lv3U-v4-v5-v6-v7-v8-v9-v10-C 1338 976 C-v6-v7-v8-v9-v10-C 981 (F) a Exon-speciflc PCR Analysis 848 C-v31-v3U-v8-v9-vl0-C 846 (G) a 781 C-v6-v7-v8-v9-C 777 The second method we used for analyz- 726 C-v8-v9-vlO-C 720 (H) ~ ing the mRNAs was amplification of the 642 C-v7-v8-v9-C 648 cDNA between primer C2A and each of 626 6 the variant exon-specific primers by ex- 511 C-v8-v9-C 516 (i) a 447 C-v3Lv3n-C 450 ponential PCR. Amplification products 423 C-v8-C or C-v3~LCc 426 were analyzed by agarose gel electro- 319 C-C 324 O) a phoresis using ethidium bromide stain- 256 b ing (Fig. 5). With this detection method 227 C'-C 232 we verified the results obtained with the Hela 728 C-v8-v9-vl0-C 720 exon-specific runoff analysis. The size of 525 C-vl0-C 528 the amplification products could not be 425 C-v8-C 426 determined as precisely as with the acry- 321 C-C 324 lamide gel electrophoresis, but for the 279 b cell lines tested, the results obtained by 229 C'-C 232 SW613 525 C-vlO-C 528 exon-specific runoff analysis were in 447 C-v31-v3n-C 450 agreement with the results obtained 423 C-v8-C or C-v3"-Cc 426 with the exon-specific PCR analysis. 320 C-C 324 A number of PCR products were se- 272 b quenced to verify the results obtained 228 C'-C 232 with the exon-specific runoff analysis. aThe letters F-J refer to the letters in Fig. 4. PCR products were isolated from low 6The composition of this minor mRNA could not be determined by exon-specific runoff analysis. melting point agarose and directly se- cUsing this technique it was not possible to determine which of these two exon combinations is quenced using cycle sequencing. We se- represented by this band. quenced the first 150 nucleotides of sev- eral of the major bands (A, C, D, E, G, I) of the HaCaT and HT29D4 cell lines. The sequence results confirmed the conclu- products. This may be caused by a differ- is, for the HeLa cell line, in agreement sions drawn from the exon-specific run- ence in CD44 expression levels in the with results reported by others. (16) In off analysis listed in Tables 2 and 3. HT29 and HT29D4 cell lines. For the these five cell lines we did not find evi- HeLa and SW613 cell line, the standard dence for a frequent use of the alterna- CD44 without variant exons is most tive acceptor site in variant exon v3 or CONCLUSION abundantly present (see also Fig. 3). This the alternative donor site in constant Several papers have been published in which it is shown that certain alterna- tively spliced forms of CD44 are ex- pressed on human metastatic tu- mors. (s'~1-13~ Because it is still unclear which exons or combination of exons are involved in the metastatic spread of tumor cells, ~ a simple method for an- alyzing CD44 expression in detail is nec- essary. Using hybridization techniques, it is laborious and difficult to determine the exact composition of each CD44 mRNA species in the cell. Antibodies against epitopes encoded by each variant exon can only be used to show the pres- ence of certain CD44 variant exon-en- coded sequences on cells but not in which context they are present in the FIGURE 5. Ethidium bromide staining of an exon-specific PCR analysis of the HaCaT (A) and protein molecule. The method described HT29D4 (B) cell lines using primer C2A and each exon-specific primer. (Top) The primers (r/ght); here is suitable for a qualitive analysis of the molecular mass marker (pSP64 • HgiAI + pSP64 • MaeI). each major CD44 mRNA in the cell. This

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method can be automated using fluores- 7. Arch, R., K. Wirth, M. Hofmann, H. Ponta, erated by insertions at a single site in the cent dye-labeled primers and sequencing S. Matzku, P. Herrlich, and M. ZOller. extracellular domain. J. Biol. Chem. 267: machines. Software can be developed for 1992. Participation in normal immune re- 4732-4739. completely automated analysis of the sponses of a metastasis-inducing splice 19. Jackson, D.G., G.R. Screaton, M.V. Bell, variant of CD44. Science 257: 682--685. and J.I. Bell. 1993. CD44 and cancer. Lan- amplification products. This would facil- 8. G~inthert, U., M. Hofmann, W. Rudy, S. cet 341: 252-253. itate screening of large numbers of hu- Reber, M. ZOller, I. Haussmann, S. man tumor samples for their CD44 com- Matzku, A. Wenzel, H. Ponta, and P. Herr- position, enabling evaluation of the lich. 1991. A new variant of glycoprotein Received May 13, 1993; accepted in relevance of CD44 splicing for diagnos- CD44 confers metastatic potential to rat revised form September 1, 1993. tic purposes. Apart from analysis of carcinoma cells. Cell 65: 13-24. CD44 mRNA, this method can also be 9. TOlg, C., M. Hofmann, P. Herrlich, and H. used in other complex splicing systems. Ponta. 1993. Splicing choice from ten variant exons establishes CD44 variabil- ity. Nucleic Acids Res. 21" 1225-1229. ACKNOWLEDGMENTS 10. Rudy, W., M. Hofmann, R. Schwartz-Al- biez, M. ZOller, K.-H. Heider, H. Ponta, We thank Fons van Mansfeld, Loesje van and P. Herrlich. 1993. The two major der Voorn, and Boudewijn Burgering for CD44 proteins expressed on a metastatic critical reading of the manuscript, Peter rat tumor cell line are derived from dif- Herrlich for discussing results before ferent splice variants: Each one individu- publication, Mark Timmers for the TBP ally suffices to confer metastatic behav- oligonucleotides, and N.E. Fusenig for ior. Cancer Res. 53: 1262-1268. the HaCaT cell line. This work was sup- 11. Tanabe, K.K., L.M. Ellis, and H. Saya. ported by a grant from the Netherlands 1993. Expression of CD44R1 adhesion Foundation for Chemical Research molecule in colon carcinomas and me- tastases. Lancet 341: 725-726. (SON), with financial support from the 12. Koopman, G., K.-H. Heider, E. Horst, G.R. Netherlands Organization for Scientific Adolf, F. van den Berg, H. Ponta, P. Herr- Research (NWO). lich, and S.T. Pals. 1993. Activated human and aggressive Non-Hodg- kin's lymphomas express a homologue of REFERENCES the rat metastasis-associated variant of 1. Screaton, G.R., M.V. Bell, D.G. Jackson, CD44. J. Exp. Med. 177: 897-904. F.B. Cornelis, U. Gerth, and J.I. Bell. 1992. 13. Matsumura, Y. and D. Tarin. 1992. Signif- Genomic structure of DNA encoding the icance of CD44 gene products for cancer lymphocyte homing receptor CD44 re- diagnosis and disease evaluation. Lancet veals at least 12 alternatively spliced ex- 340: 1053-1058. ons. Proc. Natl. Acad. Sci. 89:12160 ~ 14. Fantini, J., B. Abadie, A. Tirard, L. Remy, 12164. J.P. Ripert, A. El Battari, and J. Marvaldi. 2. Aruffo, A., I. Stamenkovic, M. Melnick, 1986. Spontaneous and induced dome C.B. Underhill, and B. Seed. 1990. CD44 is formation by two clonal cell populations the principal cell surface receptor for hy- derived from a human adenocarcinoma aluronate. Cell 61: 1303-1313. cell line HT29. J. Cell Sci. 83: 235-249. 3. Stamenkovic, I., A. 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106 PCR Methods and Applications Downloaded from genome.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press

A PCR-based method for the analysis of human CD44 splice products.

D H van Weering, P D Baas and J L Bos

Genome Res. 1993 3: 100-106 Access the most recent version at doi:10.1101/gr.3.2.100

References This article cites 19 articles, 9 of which can be accessed free at: http://genome.cshlp.org/content/3/2/100.full.html#ref-list-1

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