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[Cancer Research 60, 5529-5535, October 1, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

A Putative Oncogenic Role for MPP11 in Head and Neck Squamous Cell Cancer1

Vicente A. Resto, Otavia L. Caballero, Martin R. Buta, William H. Westra, Li Wu, Joanne M. Westendorf, Jin Jen, Philip Hieter and David Sidransky2

Departments of Molecular Biology and Genetics [V. A. R.], Otolaryngology-Head and Neck Surgery [V. A. R., O. C., M. R. B., W. H. W., L. W., J. J., D. S.], Pathology [W. H. W.], and Oncology [J. J., D. S.], The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195; Department of Molecular Pharmacology, Stanford School of Medicine, Palo Alto, California 94305 [J. M. W.]; and Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, British Columbia, V5Z 4H4 Canada [P. H.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Genetic alterations of chromosome 7 are common in human cancer. Furthermore, previous studies have supported the presence of a gene important in a broad range of cancers at 7q22–31.1. There is evidence that supports an oncogenic function for this putative gene, as well as evidence that supports a tumor suppressive role. In this study, we used a cross-species candidate gene approach in combination with physical mapping to identify MPP11 as a candidate for the putative cancer-related activity at 7q22–31.1. We then analyzed primary head and neck squamous cell tumors (HNSCCs) for loss of heterozygosity/allelic imbalance (LOH/AI) at the MPP11 genomic locus. Thirty-eight percent of tumors examined displayed LOH/AI involving the MPP11 genomic locus. Mutation analysis of MPP11 in the latter samples did not identify any inactivating mutations. However, immunohistochemical staining of primary tumor sections and Western blot analysis of HNSCC cell lines revealed a tumor-specific high level of expression of MPP11p. Fluorescence in situ hybridization analysis done on the cell lines identified increased chromosome 7 copy number with a concomitant increase in MPP11 copy number. These results suggest an oncogenic role for MPP11 in HNSCC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Genetic alterations of chromosome 7 are commonplace in human cancer (1) . LOH3 /AI of the 7q22 to 7q31 region has been reported in breast, prostate, pancreatic, ovarian, gastric, colon, and head and neck cancer, as well as uterine leiomyomas and malignant myeloid disease (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) . Furthermore, transfer of human chromosome 7 into immortalized human fibroblasts with LOH/AI at 7q31–32 restored programmed senescence to these cells (14) . In addition, experimental transfer of human chromosome 7 into murine squamous cell carcinoma cell lines induced a 2–3-fold temporal delay in the onset of most tumor explants and complete suppression in others (15) . Finally, deletions of mouse chromosome 6 have been documented in murine squamous cell cancer. Fine mapping localized these deletions to a region that is syntenic to human 7q22–31 (16) . These data support the presence of an evolutionarily conserved, broad range tumor suppressor gene at the 7q22–31 region.

In contrast to these reports, others have demonstrated genomic high-copy amplification of the 7q22–31 genomic region as an important event in the progression of prostate, gastric, germ cell, glioblastoma, and HNSCCs (17, 18, 19, 20, 21, 22, 23, 24, 25, 26) . The latter results would suggest the presence of an oncogene in this cytogenetic region.

Dysregulation of pathways related to cell cycle regulation and differentiation is a critical component of oncogenic transformation and progression (27, 28, 29) . Identifying the important molecular components of processes such as chromosome segregation and DNA replication have yielded candidate genes subsequently proven to be mutated in cancer (30) . Furthermore, many of these fundamental cellular processes, as well as their components, have often been found to be conserved throughout evolution (31) . This conservation of function supports the study of candidate genes in lower organisms for the eventual identification of genes important in human cancer (32) .

ZUO1 is such a gene in Saccharomyces cerevisiae.
It encodes a protein originally isolated as a specific Z-DNA binding protein in vitro (33) . Recent work has suggested that this gene may function as a ribosome-associated chaperone (34) . Phenotypic analysis of a null allele of ZUO1 in yeast suggested a mitogenic role for ZUO1 (33 , 34) . ZUO1 also has a mouse homologue, MIDA1, which was identified as a protein that binds to the helix-loop-helix repressive factor Id-1 (35) . Intriguingly, Id-1 was shown to be important in the negative regulation of differentiation, as well as the regulation of T antigen-dependent transformation of senescent cells (36) . Most recently, constitutive expression of Id-1 in mammary epithelium was shown to inhibit differentiation and drive invasion of the basement membrane (37) . Moreover, expression of Id-1 in adult mouse intestinal epithelium induced the development of adenomas (38) . Finally, as in yeast, phenotypic characterization of an antisense knockout in MEL cells revealed that MIDA1 acts as a mitogenic factor (35) .

We found that a novel human gene, MPP11, encoding a phosphoantigen recognized by the monoclonal MPM2, is the human orthologue of ZUO1 and MIDA1 (39) . The MPM2 antibody has been shown to recognize phosphoepitopes in centrosomes, kinetochores, spindle fibers, the chromosomal axis, and the midbody in an M-phase restricted manner, thus highlighting molecules with important functions in the mitotic phase of the cell cycle (40 , 41) .

In addition, we show that MPP11 maps to the critical region of 7q22–31.1 and is expressed in most tissues. To further characterize MPP11 as a candidate gene involved in human cancer, we performed microsatellite analysis of the critical region in paired primary HNSCC and normal DNA. AI was identified in 38% of the cases examined. Analysis of Mpp11p expression revealed high protein levels in most primary tumors and HNSCC cell lines examined. Furthermore, FISH analysis done on HNSCC lines revealed an increased copy number of the MPP11 locus in two-thirds of the lines examined. These studies support the notion that MPP11 may play an oncogenic role in HNSCC.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Gene Identification and Expression Analysis.
Conceptual translations of ZUO1 (gb X63612) and MIDA1 (gb X98260) were used as queries in the BLAST algorithm4 against the ESTdb to identify the human orthologue of these genes. The identified clones were obtained from ATCC5 and verified by direct sequencing. Expression analysis was performed using the MPP11 cDNA sequence as a query against the ESTdb. The top 20 perfect hits were evaluated for tissue of origin. The expression pattern was verified by probing a multiple tissue Northern blot (Clontech Laboratories, Inc., Palo Alto, CA) with the insert of a partial MPP11 cDNA clone (gb T11010).

Mapping.
The partial MPP11 cDNA clone hbc408 (gb T11010) was mapped via the XREFdb project (32) .6 The map location was further refined by using a YAC contig of human chromosome 7 (42) . DNA was prepared from each YAC containing strain using standard techniques for DNA purification from yeast. Primers (5'-AACAGAAGAACAGAAGCT-3' and 5'-TGCACACACAACAAAGAT-3') were used in standard PCR reactions using the purified DNAs to screen for a 523-bp fragment specific for MPP11. Positive YACs were verified by Southern blot analysis using the insert of clone hbc408 as a probe.

Intron-Exon Boundary Identification.
A human BAC clone containing the genomic MPP11 locus (gb AC004668) was identified by using the MPP11 cDNA (gb X98260) as a query in the BLAST algorithm against the nonredundant DNA database.4 Direct comparison of the MPP11 cDNA sequence against the genomic sequence identified the exons. The exon-intron boundaries were defined by examination of the genomic sequence for consensus splicing signal sequences.

DNA Extraction.
Forty-five primary HNSCCs were collected after surgical resection with prior consent from Johns Hopkins Hospital patients. Specimens were fresh frozen and microdissected on a cryostat so that tumor samples contained >70% neoplastic cells. DNA from tumor sections was digested with SDS/proteinase K, extracted with phenol-chloroform, and ethanol precipitated as described previously (43 , 44) . Normal control DNA was obtained from peripheral lymphocytes and processed in the same manner as the primary tumor samples.

Microsatellite Analysis.
DNA from tumor and normal control was examined for LOH/AI by PCR-based microsatellite analysis. Markers D7S518, D7S2446, and D7S3080 were chosen to map alterations centromeric to MPP11, whereas markers D7S501, D7S496, D7S523, and D7S486 were chosen to map alterations telomeric to MPP11. PCR conditions and criteria for AI and homozygous deletion were described previously (45) .

Sequence Analysis.
Tumors displaying AI were analyzed for mutations by direct sequencing. All 17 exons corresponding to MPP11-L were amplified and sequenced as described previously (46 , 47) . The PCR products were sequenced using the [{gamma}-33P]ATP 5' end-labeled sequencing primer and the AmpliCycle sequencing kit (Perkin Elmer, Roche Molecular Systems, Inc., Branchburg, NJ).

Immunohistochemistry.
Paraffin-embedded blocks from 10 of the 45 primary HNSCCs were obtained from archives at the Department of Surgical Pathology, The Johns Hopkins Hospital. Five-µm sections were subjected to antigen retrieval by boiling in PBS buffer for 15 s in a microwave. The samples were allowed to slowly cool to room temperature. The sections were then stained using the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). The primary antibody used was a guinea pig polyclonal antibody generated against purified Mpp11p (39) . The latter was used at a dilution of 1:1000. Finally, the Peroxidase Substrate kit DAB (Vector Laboratories, Inc.) was used to visualize the immunohistochemical stain while counterstaining with hematoxylin. All 10 cases were also stained with H&E for localization of neoplastic cells. Slides were independently reviewed and scored by a pathologist with extensive experience in head and neck neoplasms (W. H. W.).

Cell Culture.
The HNSCC cell lines used were O11, O12, O13, O19, O28, and FaDu. All of the cell lines were derived from histologically confirmed primary HNSCCs resected from patients treated at the Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins Hospital, with appropriate Institutional Review Board approval. FaDu was obtained from the ATCC.5 BEAS-2B (human bronchial epithelial cells transformed with an adenovirus 12-SV40 hybrid virus) and 1106-KERTr (skin keratinocyte, HPV-16 E6/E7 transformed) were obtained from ATCC. Cells were maintained in BEGM (Clonetics) and serum-free keratinocyte medium supplemented with recombinant human epidermal growth factor and bovine pituitary extract (Life Technologies), respectively. Primary NHBE cells and the SAECs were obtained from Clonetics and grown in appropriate media (Clonetics), according to their instructions.

FISH Analysis.
Cytocentrifuge preparations of interphase HNSCC cell line nuclei on glass slides were hybridized with a biotin-labeled, chromosome 7-specific {alpha} satellite probe (Oncor, Gaithersburg, MD) and a digoxigenin-labeled MPP 11 probe (153m3). The MPP11 probe was isolated from a BAC human genomic library (Genome Systems, Inc., St. Louis, MO), using primers that amplify exon 3 of MPP11 (5'-ACTCCTGATGAAATGTCACTT-3' and 5'-TGGATTGCTTTCTTCTGA-3'). The biotinylated probe was detected by subsequent incubation with FITC-conjugated avidin, biotin-conjugated goat antiavidin, and again FITC-conjugated avidin. The digoxigenin-labeled probe was detected by serial incubations with mouse anti-digoxigenin, rabbit antimouse-TRITC conjugate, and goat antirabbit-TRITC conjugate. Nuclei were counterstained with 1 µg/ml 4',6-diamidino-2-phenylindole, and the slides were mounted in an antifade solution and observed in an epifluorescence microscope equipped with triple-band-pass filter (4',6-diamidino-2-phenylindole, TRITC, and FITC). Images were captured with a CCD camera and processed using the Oncor Image analyzing system. Evaluation of the preparation was performed by counting at least 100 nuclei/slide. The number of MPP11 and chromosome 7 signals were counted for each nucleus. The overall mean MPP11:chromosome 7 ratio was calculated for each sample.

Western Blot Analysis.
Cell lines were grown to ~85% confluence and lysed in modified RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.01% SDS, 1 mM phenylmethylsulfonyl fluoride, and COMPLETE protease inhibitor cocktail (Boehringer Mannheim)] and then submitted to five cycles of sonication (10 s/cycle). One hundred µg of cell extract were run in 10% Tris-HCl gels and transferred onto nitrocellulose membranes (Sartorius), probed with anti-Mpp11p (39) and antiactin (Chemicon) antibodies, followed by the secondary antibodies, horseradish peroxidase-conjugated anti-guinea pig antibody and horseradish peroxidase-conjugated antimouse antibody (Pierce), respectively.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MPP11 Is the Human Orthologue of ZUO1 and MIDA1.
To find the human orthologue of ZUO1 and MIDA1, conceptual translations for both of these genes were queried against the ESTdb. The search with Mida1p identified a 95-bp human EST (clone hbc408, gb T11010) from a human pancreatic islet cDNA library. The sequence yielded a perfect match corresponding to amino acids 263–280 in the Mida1p sequence. Full sequence analysis of the insert revealed a DNA fragment of 1196 bp that predicted a protein sequence 93% identical to amino acids 255–582 of Mida1p. At about the same time, the full cDNA sequence for a novel gene of unknown function, called MPP11, was deposited in GenBank (X98260) (39) . Alignment of the hbc408 insert DNA sequence against MPP11 proved them to be identical, with the exception of a 160-bp sequence contained in hbc408 but not in MPP11. This finding suggests the presence of two alternatively spliced products herein referred to as MPP11-L (Long) and MPP11-S (Short), respectively. A final sequence comparison shows that Mpp11-Lp is 93% identical to Mida1p and 36% identical to Zuo1p (Fig. 1)Citation .



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  		Fig. 1. A, alignment of the predicted amino acid sequences for Mpp11-L, Mida1p, and Zuo1p. Perfectly conserved positions have been shaded. B, schematic representation of the motif organization for Mpp11-L (MPP11), Mida1p (MIDA1), and Zuo1p (ZUO1).

 
A more detailed analysis of the protein sequences revealed that Mpp11p shares the same motifs with both Zuo1p and Mida1p (Fig. 1)Citation . Experimental and sequence analysis data previously identified four motifs important for biological function in Zuo1p and Mida1p (34 , 35) . These are the DNA J motif, the Id-1 interaction motif, the histone H1-like motif, and the myb DNA binding motif, in NH2- to COOH-terminal orientation. These four motifs are present within the Mpp11p predicted sequence with conservation of their relative organization, further validating MPP11 as the human orthologue of MIDA1 and ZUO1.

MPP11 Is a Widely Expressed Gene.
To assess the tissue expression pattern of MPP11, the cDNA sequence was used as a BLAST query against the database of ESTs (dbEST), and the top 20 human hits were evaluated. The lowest BLAST score evaluated was 196 with an E-value of 8e-48. Four of the 20 hits were clones originating from pooled libraries; therefore, only 16 hits were scored. The cell types and tissues identified were: senescent fibroblasts (gb AI086580), HeLa cells (AA189125), germinal center B-cell (gb AA765155), colon (gb AA553955, AA524380, AA543065, and AA131089), colon cancer (AA631261), promyelocyte (gb D20011), Ewing’s sarcoma (AA639055), pregnant uterus (AA708608), testis (AI140595 and AA833629), placenta (N29844), kidney (AI244518), and aorta (D79259). To further verify the above results, a multiple tissue Northern was probed with the insert from clone hbc408. The tissues surveyed included heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. All of the tissues showed robust expression of a transcript of ~2.6 kb in size, except lung which displayed a lower level of expression (data not shown).

MPP11 Maps to 7q22–31.1.
To evaluate MPP11 as a candidate for disease phenotypes, the hbc 408 clone was submitted to the XREFdb project for mapping (32) . A Southern blot of TaqI-digested DNA from The Jackson Laboratory BSS interspecific backcross DNA panel was probed, and RFLP data were analyzed and positioned by The Jackson Laboratory. Two loci were identified by this method, one 13 cM offset on chromosome 5 and another 47 cM offset on chromosome 6. Analysis of regions of synteny between mouse and human chromosomes inferred the localization of the signals to 7q35–qter and 2pter–qter, respectively. Human chromosome localization was also performed using the same probe against DNA from a National Institute of General Medical Sciences somatic cell hybrid panel (#2, version 2). The latter analysis localized the probe to human chromosomes 3, 6, 7, and 12. Thus, the combined analysis suggested that MPP11 was located in the chromosomal region 7q35–qter.

The map location of MPP11 was further refined by mapping of the cDNA against a YAC contig of human chromosome 7 (42) . Primers were designed to amplify a 523-bp fragment at the 3' end of the gene, which included an intron. The intron was identified by sequence analysis of an incompletely spliced cDNA clone in dbEST (IMAGE consortium clone 259842, gb N29844). The YAC contig was screened by PCR amplification from purified DNA, and positive YACs were then verified by Southern blot analysis using the insert of clone hbc 408 as a probe. This analysis further refined the physical map location of MPP11 to 7q22–31.1.

Further evaluation of GenBank sequences identified a fully sequenced BAC clone RG276O03 (gb AC004668) recently deposited in the database as part of the sequencing phase of the Human Genome Project.7 The 112 Kb sequence contained the entire genomic sequence for MPP11 and was mapped to 7q22–31.1. The intron exon boundaries were defined by comparison of the cDNA sequence of MPP11 to the genomic sequence. MPP11-S was found to be comprised of 15 exons, whereas MPP11-L was comprised of 17 exons (Fig. 2)Citation .



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  		Fig. 2. Schematic representation of the genomic exon organization for splice variants MPP11-S and MPP11-L.

 
AI Occurs at the MPP11 Genomic Locus.
LOH/AI at 7q22–31 has been reported in many cancers, including HNSCC (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) . We decided to verify these results in HNSCC, with fine mapping at the MPP11 genomic locus. Examination of the genomic sequence identified a CA dinucleotide repeat located 2.1 kb upstream from the translational start site for MPP11 (D7S3080). Evaluation of the genomic sequence for chromosome 78 revealed two other microsatellite markers (D7S2446 and D7S518) located 1.3 and 3.8 Mb centromeric to D7S3080. Four microsatellite markers (D7S501, D7S496, D7S523, and D7S486) were evaluated 200 kb and 1.8, 2.9, and 8.9 Mb telomeric to D7S3080.

Forty-five paired normal/tumor DNA samples from patients with HNSCC were analyzed by microsatellite analysis at these markers. Thirty-eight percent (17 of 45) of the cases displayed LOH/AI in this region. These tumors were then further scored for LOH/AI relative to MPP11. Of the 17 tumors that showed LOH/AI, 6 cases had LOH/AI at all markers tested, 9 cases had centromeric LOH/AI including MPP11, and 2 cases had telomeric LOH/AI including MPP11 (Fig. 3)Citation . Tumors T1398, T853, T1048, T1039, and T1020 define the minimal region of loss (or imbalance) within markers D7S3080 and D7S501. Microsatellite instability was not detected in any of the cases examined.



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  		Fig. 3. Schematic representation of the patterns of LOH/AI identified in the primary tumors examined.

 
MPP11 Is Not Inactivated in Primary HNSCC.
To evaluate MPP11 as a target in the progression of HNSCC, we sequenced all 17 exons corresponding to MPP11-L in all 17 cases that displayed LOH/AI at the MPP11 locus. No inactivating mutations of the remaining allele were identified in any of the cases.

Mpp11p Is Overexpressed in HNSCC.
To distinguish between loss or gain of chromosomal material, all available paraffin sections from the primary tumors in this study (10 of 45) were analyzed by immunohistochemical staining using an antibody developed against purified Mpp11p. The subcellular localization of Mpp11p has been characterized as both nuclear and cytoplasmic (39) . Immunohistochemical staining of normal epithelium revealed staining of the germinal layer and prickle cell layer, with sparing of the granular layer and the cornified layer (Fig. 4)Citation . In contrast, examination of the tumor sections revealed that 80% (8 of 10) of the tumors stained homogeneously with what appeared to be a relative increase in staining intensity. Of the eight tumors that stained strongly for Mpp11p, 75% (6 of 8) were found to display AI in the analysis presented previously, whereas both tumors that displayed normal to absent staining also displayed no AI.



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  		Fig. 4. Immunohistochemical staining of paraffin-embedded primary tissue with a polyclonal antibody against Mpp11p. A, normal epithelium. Staining is restricted to the prickle cell layer. B, tumor 629. C, tumor 960. D, tumor 1213. x50.

 
To ascertain whether Mpp11p levels were indeed elevated in HNSCC, we evaluated the levels of Mpp11p in six HNSCC cell lines by Western blot analysis using the same anti-Mpp11p antibody referred to above. Primary cell lines SAEC (normal human) and NHBE, as well as transformed cell lines 1106-KERTr (HPV16 E6/E7 transformed normal skin keratinocytes) and BEAS-2B (SV40 transformed normal bronchial epithelium) were used as comparison controls. We found that levels of Mpp11-Lp were elevated 4–20-fold in five of six HNSCC cell lines tested as compared with 1106-KERTr and other control lines (Fig. 5)Citation . Mpp11-Sp was not expressed in any of the cell lines examined. This result, together with the immunohistochemical analysis described, suggests that Mpp11-Lp is overexpressed in HNSCC.



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  		Fig. 5. Western blot analysis of Mpp11p and actin levels in primary human bronchial epithelium (SAEC-6043 and NHBE-5607-NRA), virus transformed cell lines (1106-KERTr and BEAS-2B), and head and neck cancer cell lines (O11, O12, O13, O19, O28, and FaDu; see "Materials and Methods").

 
MPP11 Copy Number Is Increased in HNSCC Cell Lines.
To further assess the role of the MPP11 in HNSCC, we analyzed the same six cell lines for chromosome 7 and MPP11 copy number using FISH. The number of MPP11 copies detected per cell in the six cell lines examined were 3.5, 3.7, 2.2, 2.0, 5.5, and 2.9 in O11, O12, O13, O19, O28, and FaDu, respectively (Fig. 6)Citation . All four cell lines with an increase in copy number of the MPP11 locus contained a concomitant increase in the centromere signal, confirming polysomy of chromosome 7. There was no specific amplification of MPP11 and no evidence of chromosome seven deletion in any of the cell lines examined. We observed a good correlation between copy number and MPP11 expression by Western blot analysis in some cell lines (i.e., O11 and O12) but not in others (i.e., O28).



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  		Fig. 6. A, FISH on metaphase chromosomes of normal human lymphocytes with a centromeric probe for chromosome 7 (green) and a MPP 11 BAC clone probe (red) showing the MPP 11 signals in the q arm of chromosome 7. B, FISH on interphase nuclei of HNSCC cell line 012. C, FISH on interphase nuclei of HNSCC cell line 028. The green signal corresponds to the centromeric probe for chromosome 7, and the red signal corresponds to the MPP11 BAC clone probe. Both cell lines contain increased copies of chromosome 7 and MPP11. x1000.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have used the candidate gene approach to investigate a role for MPP11 in the oncogenic process of HNSCC. Cross-species sequence analysis strongly suggests that MPP11, MIDA1, and ZUO1 are true orthologues. The functional equivalence of these genes is strengthened by the fact that an antisense knockout of MIDA1 in MEL cells has an identical phenotype to that seen in a yeast zuo1 null strain (33, 34, 35) . Furthermore, the immunofluorescent subcellular localization of Zuo1p is identical to that reported for Mpp11p (39) .9

Although the function of MPP11 is not known, this gene has been conserved throughout evolution, suggesting a similar function to that of its orthologues. MIDA1 interacts with Id-1 and may regulate its function, and Id-1 has been implicated in the processes of cellular differentiation and cell invasion (35 , 37) . Furthermore, a direct link between Id-1 and SV40 T antigen transformation has been shown (36) . ZUO1 may encode a ribosome-associated chaperone in yeast; however, it was first isolated as a gene encoding a DNA binding protein (33 , 34) . Most recently, ZUO1 has been implicated in the transcriptional regulation of CTF13, a gene encoding an essential kinetochore component in yeast and aberrations in the kinetochore function lead to chromosome missegregation (48) .9 Therefore, it is interesting that Mpp11p is identified by the MPM2 antibody, which also identifies multiple phosphoepitopes important in mitotically related structures (39) . These results suggest a regulatory role for MPP11 in the mitotic phase of the cell cycle, perhaps in the regulation of kinetochore function. Chromosome segregation, differentiation, and cell invasion are all processes that are intricately involved in the genesis and progression of human cancer; thus, MPP11 is a strong candidate gene in cancer.

A complete allelotype of HNSCC has identified LOH on chromosomes 3, 5, 9, 11, 13, and 17 occurring in 45% or more of cases examined (49 , 50) . The high frequency loss of chromosomes 3, 9, and 17 in early preinvasive lesions suggests these changes may occur early in the progression of HNSCC (44) . LOH at 7q has been reported to occur in 30% of HNSCC, and finer mapping at 7q31.1 has revealed LOH in >50% of cases (12 , 49) . On the basis of Knudson’s hypothesis, we searched for mutations of the remaining allele in tumors with LOH at the MPP11 locus. We found no inactivating mutations. This result rejected the notion of a tumor suppressive role for MPP11. However, the same region was shown to be amplified in as many as 30% of HNSCC (17) . This apparent difference is reconciled by comparison of FISH analysis with the LOH/AI data. LOH is usually assumed to represent a relative loss or deletion of chromosomal DNA at a particular marker. However, chromosomal gains can manifest as a relative change in gene dosage or imbalance when compared with a normal control sample. FISH analysis reveals an increased copy number for this region, accompanied by chromosome 7 polysomy.

The latter observation is most consistent with the biological role of MPP11 as a mitogenic factor (33, 34, 35) . Our immunohistochemical analysis of primary HNSCC tumors revealed that Mpp11p was highly expressed in 80% of the cases examined. When normal epithelium was examined, Mpp11p expression was found to be restricted to the germinal and prickle cell layers. This result is of interest because these epidermal cell layers are the only epidermal histological layers that display cell division, a characteristic shared with cancerous cells. Furthermore, analysis of Mpp11p expression in HNSCC cell lines also showed an increased level of Mpp11-Lp expression relative to normal epithelial cell lines, as well as transformed epithelial cell lines, thus suggesting that the increase in Mpp11-Lp expression is not solely a marker of cell division. Mpp11-Sp expression was not detected in any of the epithelial cell lines examined, thus suggesting it is not important in the biology of normal or malignant epithelial cells.

These increases in Mpp11-Lp expression level appear to be attributable in part to increased copy number in both primary tumors (AI) and cell lines (increased copy number by FISH). Other mechanisms, such as increased transcription, probably contribute to changes in expression that are independent of genomic dosage, as was seen in several of our cell lines. Notwithstanding, these results lend support for a tumor-specific increase in Mpp11-Lp expression. The question remains as to whether the high expression Mpp11-Lp is an important change in the progression of cancer or simply a marker of tumorigenesis.

Increased copy number of MPP11 and overexpression suggest that this gene is activated during cancer progression. This notion is certainly consistent with the fact that many cancers become highly aneuploid and invasive at more advanced stages. Alternatively, another oncogene may reside near MPP11, within the minimally amplified region. A better understanding of the role played by MPP11 in human cancer awaits further characterization of the gene in other tumor types and additional studies that assess the ability of MPP11 to transform cells in vitro and in vivo.


   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 This work was supported by National Institute of Dental and Craniofacial Research grant RO1-DE012588–01 (to D. S.). V. A. R. is supported by National Institute of General Medical Sciences Predoctoral Fellowship Grant GM 18041. O. L. C. is supported by Fellowship 1998/2736-2 from the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil. Back

2 To whom requests for reprints should be addressed, at Department of Otolaryngology-HNS, Division of Head and Neck Cancer Research, The Johns Hopkins University School of Medicine, 818 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21206-2198. E-mail: dsidrans{at}jhmi.edu Back

3 The abbreviations used are: LOH, loss of heterozygosity; AI, allelic imbalance; HNSCC, head and neck squamous cell carcinoma; FISH, fluorescence in situ hybridization; YAC, yeast artificial chromosome; BAC, bacterial artificial chromosome; ATCC, American Type Culture Collection; BEGM, bronchial epithelial basal and growth medium; NHBE, normal human bronchial epithelium; SAEC, small airway epithelial cell; TRITC, tetramethyl rhodamine isothiocyanate; EST, expressed sequence tag. Back

4 Internet address: http://www.ncbi.nlm.nih.gov/BLAST/. Back

5 Internet address: http://www.atcc.org. Back

6 Internet address: http://www.ncbi.nlm.nih.gov/XREFdb/. Back

7 T. Rohlfing et al., direct submission. Back

8 The Genome Database, http://gdbwww.gdb.org/. Back

9 V. A. Resto, J. L. Corden, and P. Hieter, manuscript in preparation. Back

Received 1/ 3/00. Accepted 8/ 1/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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