Chromosomal instability and cytoskeletal defects in oral cancer cells William S. Saunders*†, Michele Shuster‡, Xin Huang‡, Burhan Gharaibeh‡, Akon H. Enyenihi*, Iver Petersen§, and Susanne M. Gollin‡ Departments of *Biological Sciences and ‡Human Genetics, University of Pittsburgh and the University of Pittsburgh Cancer Institute, Pittsburgh, PA 15260; and §Institute of Pathology, University-Hospital Charite, D-10098 Berlin, Germany Edited by Janet D. Rowley, The University of Chicago Medical Center, Chicago, IL, and approved October 25, 1999 (received for review July 1, 1999) Oral squamous cell carcinomas are characterized by complex, often somal changes, such as breakpoints at bands 1p11–1p13, 3p14, near-triploid karyotypes with structural and numerical variations and 11q13 (6) and amplification of band 11q13 (7, 8). Chromo- superimposed on the initial clonal chromosomal alterations. We somal rearrangements may be driven andyor fixed by activation used immunohistochemistry combined with classical cytogenetic of oncogenes or loss of tumor suppressor genes. For example, the analysis and spectral karyotyping to investigate the chromosomal 11q13 amplicon includes CCND1, the cyclin D1 gene, which segregation defects in cultured oral squamous cell carcinoma cells. plays a role in promoting cell division (9). During division, these cells frequently exhibit lagging chromo- We have begun to investigate the source of chromosomal somes at both metaphase and anaphase, suggesting defects in the instability in cultured cells derived from human OSCC. We have mitotic apparatus or kinetochore. Dicentric anaphase chromatin found high levels of both numerical and structural chromosome bridges and structurally altered chromosomes with consistent long variations, including marker chromosomes and dicentrics. Dur- arms and variable short arms, as well as the presence of gene ing mitotic division, we observed multiple segregational defects, amplification, suggested the occurrence of breakage–fusion– including multipolar spindles, lagging chromosomes, and an- bridge cycles. Some anaphase bridges were observed to persist aphase bridges, leading to chromosomal breakage at telophase into telophase, resulting in chromosomal exclusion from the re- into centric and acentric micronuclei. In some cases, the an- forming nucleus and micronucleus formation. Multipolar spindles aphase bridges contained DNA from chromosome 11. An ad- were found to various degrees in the oral squamous cell carcinoma ditional observed defect was the untimely and unnatural division lines. In the multipolar spindles, the poles demonstrated different of the nuclear mitotic apparatus protein NuMA at the centro- levels of chromosomal capture and alignment, indicating func- somes, apparently an early step in the splitting of the spindle tional differences between the poles. Some spindle poles showed poles. These observations support the conclusion that many of premature splitting of centrosomal material, a precursor to full the cell-to-cell variations in chromosome structure and numbers separation of the microtubule organizing centers. These results in cancer cells result from (i) dicentric chromosomes forming indicate that some of the chromosomal instability observed within anaphase bridges, which break and reform similar to that these cancer cells might be the result of cytoskeletal defects and observed originally in maize by Barbara McClintock (10) and (ii) breakage–fusion–bridge cycles. specific structural defects in the spindle and chromosome seg- MEDICAL SCIENCES regational machinery. enetic instability or changes in chromosome structure and Gnumbers is an important facet of oncogenesis. The conse- Materials and Methods quence of genetic instability can be an alteration in copy number Cell Culture. In this study, we examined three of our OSCC cell of one or more genes, a change in gene expression, or a change lines, UPCI:SCC003, UPCI:SCC131, and UPCI:SCC172, un- in gene structure such that the protein sequence is altered (1). cloned outgrowths developed from primary tumors from con- These genetic changes can lead to either increased or diminished senting patients who had not been treated previously with protein activity or can create a new gain-of-function activity for chemotherapy or radiation therapy (S.M.G., J. K. Reddy, S. the altered protein. Genetic instability can result from changes Comsa, K. M. Rossie, C. M. Lese, M. S., B. N. Appel, R. Wagner, in chromosome structure, through errors in DNA metabolism, E. N. Myers, and J. T. Johnson, unpublished work). The cells repair, recombination, or other rearrangements of the DNA were cultured in MEM with Earle’s salts supplemented with sequence (2), or from misregulation of the cell cycle, for nonessential amino acids (Life Technologies, Grand Island, example, uncoupling DNA replication from cell division (3) or NY), L-glutamine, gentamicin, and 10% FBS (Irvine Scientific). centrosomal duplication from division (4). Abnormalities in the chromosomal segregational apparatus are also likely to play an Classical Cytogenetic Analysis. The cell cultures were harvested by important role in genetic instability. These include centrosomal using standard cytogenetic techniques (11). Metaphase cells defects, defects in kinetochore–microtubule attachment, and from all three cell lines were digitally imaged and karyotyped movement of chromosomes relative to the poles. While it is likely after trypsin-Giemsa banding (7) with a CytoVision Ultra Sys- that all of these changes contribute to carcinogenesis, the extent tem (Applied Imaging, Santa Clara, CA). Karyotypes have been to which each plays a part is still largely uncharacterized. or will be published elsewhere (ref. 7, and C. M. Lese, T. Ried, One of the most striking manifestations of genetic instability in cancer cells is the variation observed in the karyotypes of different cells, even within the same tumor. A well-characterized This paper was submitted directly (Track II) to the PNAS office. example is squamous cell carcinomas of the oral cavity. The Abbreviations: OSCC, oral squamous cell carcinomas; DAPI, 49,6-diamidino-2-phenylindole; karyotypes of oral squamous cell carcinomas (OSCC) are com- SKY, spectral karyotyping; MTOC, microtubule organizing center; BFB, breakage–fusion– plex, often near triploid, and are composed of multiple numer- bridge; FISH, fluorescence in situ hybridization; NuMA, nuclear mitotic apparatus protein; CREST, calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and ical and structural abnormalities, including deletions, balanced telangiectasias. and unbalanced translocations, isochromosomes, dicentric chro- †To whom reprint requests should be addressed. E-mail: [email protected]. mosomes, and homogeneously staining regions (5). Despite the The publication costs of this article were defrayed in part by page charge payment. This numerous and diverse abnormalities, classical cytogenetic anal- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. yses of OSCC cells have revealed several consistent chromo- §1734 solely to indicate this fact. PNAS u January 4, 2000 u vol. 97 u no. 1 u 303–308 Downloaded by guest on October 1, 2021 Fig. 1. (A) Display image of metaphase chromosomes from a Colcemid-arrested UPCI:SCC172 cell after SKY analysis. Arrowhead identifies the marker chromosome ‘‘c’’ shown in B.(B) Normal chromosomes 3, 4, 8, and 11 from this culture are shown. Marker chromosomes, also from UPCI:SCC172, are indicated as a, b, and c. (C) A representative dicentric chromosome is shown from a Colcemid-arrested UPCI:SCC131 cell C-banded to highlight the centromeres. W. Gottberg, J. W. Wilson, S. C. Reshmi, J. K. Reddy, J. T. Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, Johnson, E. N. Myers, and S.M.G., unpublished work). To and telangiectasias) at 1:80 or undiluted mouse monoclonal examine the number of centromeres per chromosome, the anti-tubulin. The coverslip with primary Ab was placed in a metaphase cells were stained with C-banding using barium humid chamber at 37°C for 1 hr and then washed several times hydroxide and Giemsa stain (12). with PBSy1% BSA. The secondary Abs were Cy3-conjugated sheep anti-mouse IgG (Sigma) used at 1:1,000, FITC-conjugated Chromosome 11 Painting. For chromosome 11 painting of cells goat anti-mouse (Sigma) at 1:100, or FITC-conjugated goat growing in situ on Lab-Tek chamber slides (Nalge Nunc, Na- anti-human IgG at 1:100 (Roche Molecular Biochemicals). In all perville, IL), the cell culture medium was removed by aspiration, cases, the coverslips were counterstained with DAPI at 1–3 and the cells were fixed in 3:1 methanol:acetic acid for 100 min, mgyml. rinsed several times with 70% acetic acid solution, and air dried. The slides were aged for 30 min in 23 SCC at 37°C, pretreated Results with RNase for 1 hr at 37°C, then with pepsin for 10 min at 37°C, For these analyses, we selected three of our OSCC cell lines, followed by postfixation in 1% formaldehyde for 7 min. Slides designated UPCI:SCC003, UPCI:SCC131, and UPCI:SCC172. were denatured for 2 min at 70°C in 70% formamidey23 SSC, The TP53 gene (exons 5–8) in all three tumors and derived cell then dehydrated and air-dried prior to hybridization. The Spec- lines was sequenced and was found to be wild type. The tumor trum Green directly labeled WCP11 probe (Vysis, Downers tissue was negative for human papillomavirus by the PCR. To Grove, IL) was hybridized according to the manufacturer’s examine the variation