Oral squamous cell carcinomas are characterized by complex, often near-triploid karyotypes with structural and numerical variations superimposed on the initial clonal chromosomal alterations. We used immunohistochemistry combined with classical cytogenetic analysis and spectral karyotyping to investigate the chromosomal segregation defects in cultured oral squamous cell carcinoma cells. During division, these cells frequently exhibit lagging chromosomes at both metaphase and anaphase, suggesting defects in the mitotic apparatus or kinetochore. Dicentric anaphase chromatin bridges and structurally altered chromosomes with consistent long arms and variable short arms, as well as the presence of gene amplification, suggested the occurrence of breakage-fusionbridge cycles. Some anaphase bridges were observed to persist into telophase, resulting in chromosomal exclusion from the reforming nucleus and micronucleus formation. Multipolar spindles were found to various degrees in the oral squamous cell carcinoma lines. In the multipolar spindles, the poles demonstrated different levels of chromosomal capture and alignment, indicating functional differences between the poles. Some spindle poles showed premature splitting of centrosomal material, a precursor to full separation of the microtubule organizing centers. These results indicate that some of the chromosomal instability observed within these cancer cells might be the result of cytoskeletal defects and breakage-fusion-bridge cycles.

To determine whether the FHIT gene at 3pl4.2 is altered in head and neck squamous cell carcinomas (HNSCC), we examined 26 HNSCC cell lines for deletions within the FHIT locus by Southern analysis, for allelic losses of specific exons FHIT by fluorescence in situ hybridization (FISH) and for integrity of FHIT transcripts. Three cell lines exhibited homozygous deletions within the FHIT gene, 55% (15/25) showed the presence of aberrant transcripts, and 65% (13/20) Head and neck cancers represent 3% of all cancer in Western countries (1), and in some geographical regions such as India, the incidence is as high as 45% (2); 90-95% of these tumors are head and neck squamous cell carcinomas (HNSCC). HNSCC has a high mortality rate with a 5 year survival of 40%. Tobacco and alcohol have been recognized as etiological factors of these carcinomas, and the reported increase in the incidence of HNSCC by epidemiological studies is probably due to changes in consumption of these agents (2).Several regions of loss of heterozygosity (LOH) have been identified in HNSCC recently, including regions of 3p, 9p, llq, 13q, and 17p (3, 4). Among these loci, the 9p region presents the highest rate of genetic alteration at 75%, followed by alterations of 3p ranging between 45% and 55% (4, 5). LOH of 9p has been linked to the tumor suppressor gene, CDKN2 (6-8), whereas LOH on 3p has not yet been associated with specific genes.Deletions of the short arm of chromosome 3 are common in many human cancers, including sporadic and hereditary renal carcinomas, small-cell lung carcinomas (SCLC) (9, 10) and non-small-cell lung carcinomas (NSCLC) (11, 12) and carcinomas of the breast and cervix (13,14). In this regard, it is interesting to note that the most common fragile site in humans, FRA3B, is at 3pl4.2 (for reviews, see refs. 15 Recently, we have cloned the FHIT gene, at 3pl4.2, which encompasses the FRA3B fragile site, is disrupted by the t(3;8) chromosomal translocation observed in a family with renal cell carcinoma, and spans a region commonly deleted in cancer cell lines (20,21). We have also shown that "80% of SCLC and 40% of NSCLC express aberrant FHIT transcripts (22), suggesting that the FHIT gene is a frequent target for alteration in lung tumors. SCLC and HNSCC present the same type of histology, share major etiological factors such as tobacco (23,24), and exhibit similar regions of LOH of the short arm of chromosome 3. To determine whether the FHIT gene is also disrupted and aberrantly transcribed in HNSCCs, we studied 26 early passage HNSCC-derived cell lines. MATERIALS AND METHODS RNA Extraction and Reverse Transcription-PCR (RT-PCR). Total RNA was extracted from cell lines using RNAzol (Tel-Test, Friendswood, TX) and cDNA synthesized from 1 ,ug of total RNA. RT was performed in a 20 ,ul volume of 1 x first strand buffer (GIBCO), 10 mM DTT, 500 mM of each dNTP, 0.3 mg/ml random primers (GIBCO), and 300 units of SuperScript II (GIBCO) reverse transcriptase. The samples were first incubated 5 min at 65°C and then at 37°C ...

With the advent of wireless technology, new tools are available that are intended to enhance students' learning and attitudes. To assess the effectiveness of wireless student response systems in the biology curriculum at New Mexico State University, a combined study of student attitudes and performance was undertaken. A survey of students in six biology courses showed that strong majorities of students had favorable overall impressions of the use of student response systems and also thought that the technology improved their interest in the course, attendance, and understanding of course content. Students in lower-division courses had more strongly positive overall impressions than did students in upper-division courses. To assess the effects of the response systems on student learning, the number of in-class questions was varied within each course throughout the semester. Students' performance was compared on exam questions derived from lectures with low, medium, or high numbers of in-class questions. Increased use of the response systems in lecture had a positive influence on students' performance on exam questions across all six biology courses. Students not only have favorable opinions about the use of student response systems, increased use of these systems increases student learning.

The purpose of this study was to generate stable cell cultures from head and neck squamous cell carcinomas (HNSCC), and retrospectively analyze the factors associated with successful cell line establishment. Fifty-two HNSCC cell lines were isolated from a series of 199 tumors collected between 1992 and 1997 at the University of Pittsburgh Medical Center. Cell lines were characterized at the molecular and cellular level to determine the features associated with cell line formation. Successful cell line formation was dependent on multiple factors, including gene amplification involving chromosomal band 11q13, local and/or regional involvement of lymph nodes, and alcohol usage. The establishment of HNSCC cell lines enriches the resources available for cancer research. Our findings indicate that generation of stable cell lines from HNSCC is biased towards tumors with a poor prognosis. Our 52 stable lines comprise one of the largest series of HNSCC cell lines in the literature, with complete demographic, histopathologic, clinical, and survival data.

Gene amplification is a common feature of tumors. Overexpression of some amplified genes plays a role in tumor progression. Gene amplification can occur either extrachromosomally as double-minute chromosomes (dmin) or intrachromosomally in the form of homogeneously staining regions (hsrs). Approximately one-half of our oral squamous cell carcinomas (OSCCs) are characterized by amplification of band 11q13, usually as an hsr located entopically (occurring or situated at the normal chromosomal site, as opposed to ectopically). Using chromosomal fluorescence in situ hybridization (FISH), we confirmed the amplification of the cyclin D1 (CCND1/PRAD1) and fibroblast growth factor types 3 and 4 (FGF3/INT2 and FGF4/HSTF1) genes within the 11q13 amplicon in our series of primary OSCCs and derived cell lines. The human RIN1 gene was isolated as an RAS interaction/interference protein in a genetic selection in yeast and has been described as a putative effector of both the RAS and ABL oncogenes. We mapped RIN1 to 11q13.2. FISH analysis of 10 11q13-amplified OSCC cell lines revealed high-level RIN1 amplification in two cell lines. Three additional cell lines have what appear to be duplications and/or low-level amplification of RIN1, visible in both interphase and metaphase cells. The hybridization pattern of RIN1 on the metaphase chromosomes is particularly revealing; RIN1 signals flank the 11q13 hsr, possibly as a result of an inverted duplication. The gene amplification model of Coquelle et al. (1997) predicted that gene amplification occurs by breakage-fusion-bridge (BFB) cycles involving fragile sites. Our data suggest that the pattern of gene amplification at 11q13 in OSCC cell lines is consistent with a BFB model. RIN1 appears to be a valuable probe for investigating the process of gene amplification in general and, specifically, 11q13 amplification in oral cancer.