Transcript map of the 8p23 putative tumor suppressor region.

Cancers of the head and neck, prostate, liver, and bladder exhibit minimal regions of deletion within chromosomal band 8p23 that either overlap or map very close to one another. We previously refined a minimal region of deletion in squamous cell carcinomas to a 112-kb interval within 8p23. There seems to be only a single gene within this region that is expressed in normal upper aerodigestive tract epithelium. This candidate for the squamous cancer suppressor, CUB and sushi multiple domains-1 (CSMD1), extends into the minimal regions of deletions defined for the other types of cancer with 8p23 deletions. RT-PCR and EST data indicate that CSMD1 is also expressed in those organs,making this gene a candidate for a suppressor of multiple types of cancer. Both the sequence of the gene and the organization of the protein are highly conserved in the mouse.

Homozygous deletions define a region of 8p23.2 containing a putative tumor suppressor gene.

Loss of heterozygosity at microsatellite loci in chromosomal band 8p23.2 is a frequent event in squamous cell carcinomas of the head and neck, suggesting that this region contains a putative tumor suppressor. Allelic loss studies on laryngeal and oral/oropharyngeal tumors have restricted the size of this region to approximately 1 cM. A similar pattern of deletions is also observed in prostatic and ovarian adenocarcinomas. As part of an effort to identify this gene by positional cloning, we developed a physical contig consisting of 12 overlapping bacterial artificial chromosome (BAC) clones spanning this interval. We developed sequence-tagged sites from the ends of these BACs and used them, along with seven microsatellite loci, to detect and map homozygous deletions in four head and neck squamous cancer cell lines. Our mapping analysis further restricted the consensus minimal region of deletion to a <191-kb interval.

Multiple regions of deletion on chromosome arm 13q in head-and-neck squamous-cell carcinoma.

Several lines of evidence suggest that the progression of head-and-neck squamous-cell carcinoma (HNSCC) involves inactivation of at least one and possibly several tumor-suppressor genes on the long arm of chromosome 13. The fact that neither Rb1 nor BRCA2 appears to be inactivated in the majority of head-and-neck cancers suggests that novel tumor-suppressor genes are involved. We have used microsatellite repeat polymorphisms and PCR to detect several distinct minimal regions of deletion on 13q in supraglottic and oral squamous-cell carcinomas. One region maps to 13q34, the second to 13q14.3 and a potential third region, not reported in previous studies, maps to 13q12.1. Overall, 69% of the 145 tumors examined demonstrated allelic loss at one or more loci on 13q. We investigated whether a novel suppressor candidate mapping to 13q14. 3-q21, leukemia-associated gene 1, might also be involved in the progression of squamous-cell carcinomas. Multiplexed PCR revealed homozygous deletion of leu1 in one oral cavity tumor. This suggests that this gene or one nearby may be the actual target of deletions in this region of the chromosome arm.

Localization of a putative tumor suppressor gene in the sub-telomeric region of chromosome 8p.

Several regions of chromosome arm 8p are frequently deleted in a variety of human malignancies including those of the prostate, head and neck, lung, and colon, suggesting that there is more than one tumor suppressor gene on this chromosome arm. Both laryngeal and oral squamous cell carcinomas exhibit three distinct and nonoverlapping regions of deletion on 8p. We have further refined the localization of the putative suppressor in 8p23 by using eight microsatellite loci to create a high resolution deletion map of 150 squamous cell carcinomas of the larynx and oral cavity. These new data demonstrate that there are two distinct classes of deletion within this relatively small region of the chromosome and suggest two possible locations for the gene within the D8S264 to D8S1788 interval. We also determined that there is little difference between the allelic loss frequencies of microsatellites mapping near the telomeric ends of other chromosome arms and loci mapping to more centromere proximal regions of the same arm. These data suggest that the high allelic loss frequencies seen at 8p23 loci are not the result of a generalized instability of chromosome ends and are instead consistent with the activation of a specific suppressor gene.

Clinical correlations with allelotype in supraglottic squamous cancer.

Frequent allelic loss at a genetically polymorphic locus in tumors is an established marker for the presence of a tumor suppressor gene in the neighboring chromosomal region. This technique can be used to identify novel tumor suppressor genes and to monitor their status before the cloning of the gene itself. We have used the polymerase chain reaction and microsatellite loci on all 39 nonacrocentric autosomal chromosomal arms to identify sites of frequent allelic loss in squamous cell carcinomas of the supraglottic larynx. Our allelotype identified seven chromosomal arms (3p, 5q, 8p, 9p, 9q, 13q, and 17p) likely to contain tumor suppressor genes frequently inactivated during squamous tumorigenesis in the larynx. We tested for associations between allelic losses on these chromosomal arms and the clinical and histopathologic features of these tumors. There were no correlations with either T or N classifications. Allelic loss on chromosomal arm 13q is significantly associated with a number of histopathologic features characteristic of poorly differentiated or histologically aggressive tumors. Allelic loss on this arm also exhibits statistical trends toward association with early tumor recurrence and poor survival. The association with survival was substantiated by a multivariate Cox proportional hazards model.

Chromosome 8 allelic loss and the outcome of patients with squamous cell carcinoma of the supraglottic larynx.

BACKGROUND: Loss of genetic heterogeneity (allelic loss or loss of heterozygosity) on chromosome arm 8p is frequent in squamous cell carcinomas of the head and neck and has been associated with poor prognosis. We have previously demonstrated that there are three minimal regions of allelic loss on this chromosome arm. The location of each region is marked by a microsatellite locus: D8S264 (8p23), D8S552 (8p23-p22), and D8S133 (8p21). These findings imply the existence of at least three putative tumor suppressor genes on this chromosome arm that may become inactivated during the progression of squamous cell carcinoma. PURPOSE: We used allelic loss data from these three loci to determine if inactivation of these putative suppressors is associated with poor prognosis for patients with squamous cell carcinoma of the supraglottic larynx. We also used multivariate statistics to compare the prognostic power of allelic loss at these genetic markers with that of demographic, clinical, and histopathologic parameters. METHODS: We examined the D8S264, D8S552, and D8S133 microsatellites in tumors from a retrospective population of 59 patients. All patients had histologically confirmed squamous cell carcinoma of the supraglottic larynx and had been treated surgically. DNA was extracted from matched sets of normal and microdissected tumor tissue and used for polymerase chain reaction amplification of the microsatellite markers. Reaction products were separated by denaturing gel electrophoresis and visualized by autoradiography. Patient data were obtained from the original pathology report and from the tumor registry of the Department of Otolaryngology-Head and Neck Surgery, Washington University School of Medicine, St. Louis, MO. Histopathologic data were obtained by reviewing the portion of the resection specimen used for DNA extraction. Parameters whose association with reduced disease-free interval and reduced disease-specific survival was statistically significant were identified by use of the Kaplan-Meier method and the logrank statistic. Multivariate Cox proportional hazards models were used to identify independent predictors of poor prognosis. All statistical tests were two-sided. RESULTS: In this patient population, allelic loss at the D8S264 locus was associated with both shorter disease-free interval (logrank P = .028) and reduced disease-specific survival (logrank P = .004). Allelic loss at the next most centromeric locus, D8S552, had a statistically significant association with only reduced disease-specific survival (logrank P = .034), whereas allelic loss at the most centromeric region, D8S133, showed no statistically significant association with reductions in either interval. Multivariate Cox models suggested that D8S264 was the only 8p marker of the three microsatellites with a statistically significant and independent association with shortened disease-free interval (relative risk [RR] = 3.38; P = .0107) and reduced disease-specific survival (RR = 3.41; P = .0105). CONCLUSIONS: Allelic loss in the p23 region of chromosome 8 appears to be a statistically significant, independent predictor of poor prognosis in patients with supraglottic squamous cell carcinoma.

Evidence for multiple tumor suppressor genes on chromosome arm 8p in supraglottic laryngeal cancer.

Loss of heterozygosity studies of a variety of human tumors suggest that there are several tumor suppressor genes on chromosome arm 8p. To localize these genes more precisely, we utilized polymerase chain reaction amplification of microsatellite repeat polymorphisms and examined the allelic loss patterns of 17 marker loci on 8p in a population of 59 supraglottic laryngeal squamous cell carcinomas. Twenty-three of these tumors (39%) had an allelic loss at one or more of the markers examined. The allelic loss patterns of these tumors support the presence of at least three different tumor suppressor genes on 8p: one in 8p23, one in 8p22-23, and another in 8p21.

Allelic loss in squamous cell carcinomas of the larynx: discordance between primary and metastatic tumors.

The mutational inactivation of suppressor genes, a process required for cancer progression, generates new genetic subclones within a tumor. The allelic losses that frequently unmask these mutations serve not only as markers of the chromosomal locations of these genes but also as clonal fingerprints of the shifting relationships between these genetically heterogeneous cell populations. The rise of the metastasis-competent subclone to dominance within the primary tumor should be reflected in the similarity of the genetic fingerprints of the primary tumor and its resultant metastases. We have tested this hypothesis by comparing the patterns of allelic loss of individual primary laryngeal squamous cell carcinomas and their resultant cervical lymph node metastases at 16 different genetically polymorphic loci on 15 chromosome arms. Although primary tumors and metastases both frequently lose heterozygosity on the same chromosome arms (3p, 9p, 9q, 13q, and 17p), five of the 12 metastases differed from their primary tumors at one or two of the loci examined. Discordance between the two tumor cell populations from the same patient is suggestive of either subclone heterogeneity within the primary tumor at the time of establishment of the metastasis or further clonal evolution of both tumors after metastasis.

Frequent loss of heterozygosity for Rb, TP53, and chromosome arm 3p, but not NME1 in squamous cell carcinomas of the supraglottic larynx.

BACKGROUND: The inactivation of some tumor suppressor genes classically manifests itself through the loss of heterozygosity at nearby genetic mapping markers. Inactivation of these genes appears to have diagnostic/prognostic significance in some types of tumors. Molecular genetic tools based on suppressor inactivation might, therefore, have great utility in treatment planning. METHODS: The polymerase chain reaction and highly informative microsatellite markers were used to compare DNA derived from matched sets of tumor and normal tissue samples from 37 supraglottic laryngeal squamous cell carcinomas. Tumor samples were microdissected free of contaminating normal tissue to maximize the detection of allelic loss. Polymerase chain reaction products were fractionated by denaturing gel electrophoresis and were visualized by autoradiography. RESULTS: Allelic losses were frequent at TP53 (56% of the tumors), the retinoblastoma gene (Rb, 59%), and the p13-14 region of chromosome 3 (64%). In contrast, the putative metastasis suppressor, NME1 (also known as NM23), was lost infrequently (7%). NME1 allelic loss did not correlate with the presence of lymph node metastases in these patients. CONCLUSIONS: The high frequencies of allelic loss at TP53, Rb, and 3p13-14 suggest that these suppressors play a major role in laryngeal carcinogenesis. In sharp contrast, the low frequency of loss at NME1 and its equal distribution in nodal metastasis-positive and -negative patients suggests that inactivation of this gene by allelic loss probably does not play a role in the development of regional metastases from these tumors. Allelic loss in 3p13-14 was found in tumors of all histopathologic grades.

Repression and activation of the Drosophila dopa decarboxylase gene in glia.

Glial expression of the Drosophila dopa decarboxylase gene (Ddc) is repressed by a regulatory region located approximately 1 kb upstream of the transcriptional start site. We have used in vitro mutagenesis and germ line transformation to determine which elements within the Ddc promoter mediate repression. Our evidence suggests that the hypodermal cell activator elements IIA and IIB play a major role in the transcriptional regulation of Ddc in glial cells. A variety of mutations demonstrate that element IIA is a strong glial activator element and that element IIB is necessary for glial repression. Although these two regulatory elements are nearly identical in sequence, our data suggest that they are not redundant. Altering the wild-type number and spacing of elements IIA and IIB indicates that the wild-type arrangement of this repeat is critical for repression. We conclude that these key elements of the Ddc promoter regulate both activation and repression in glia.

Mutations within the Ddc promoter alter its neuron-specific pattern of expression.

The Drosophila dopa decarboxylase gene (Ddc) is expressed in a reproducible set of approximately 150 neurons, and in a subset of the glia of the third instar larva's central nervous system (CNS). Expression in this pattern requires a cell type-specific neuronal enhancer/glial repressor region located 1000 bp from the transcriptional start site, and specific sequences within the promoter. We have used mutagenesis in vitro and P-element-mediated transformation to examine the role of the promoter, particularly its major CNS activator sequence (element I), in the generation of the wildtype expression pattern. Immunohistological analysis of these transgenic strains demonstrates that particular deletion mutations shift the site of transgene expression to a set of neurons which do not express Ddc at detectable levels in wild-type larvae. Transgene expression in these strains may be driven by a previously undetected activator sequence. Our data also suggest that glial expression may be driven by the same activator sequences that drive expression in the hypoderm.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

CNS and hypoderm regulatory elements of the Drosophila melanogaster dopa decarboxylase gene.

Expression of the dopa decarboxylase gene (Ddc) is regulated in a tissue- and developmental stage-specific manner throughout the life cycle of the fruit fly, Drosophila melanogaster. Essential Ddc regulatory elements lie within 208 base pairs upstream from the RNA start point. Functional elements within this 5' flanking region were mapped by deletion analysis, which assayed expression in vivo after germline integration via P element vectors. One of the elements is essential for expression in both the larval and adult central nervous system, and at least two other elements are necessary for quantitatively normal expression in the hypoderm. Within each of the intervals that have regulatory effects are found sequence elements conserved between the Ddc genes of two distantly related species of flies. On the basis of this correlation, regulatory functions for these sequence elements can be postulated.

The cloned dopa decarboxylase gene is developmentally regulated when reintegrated into the Drosophila genome.

The Drosophila dopa decarboxylase gene, Ddc, functions normally when reintroduced into flies. DNA containing a cloned Ddc gene inserted into a P element transposon was injected into early embryos. Transformants were identified by suppression of the cuticular phenotype of a Ddc mutant allele. The reintegrated genes are expressed in the proper tissue and at the proper stages during development even though their positions within the genome are different from that of the wild-type Ddc gene. Absolute levels of DDC enzyme activity are within 35% of that found in wild-type Canton S flies, the source of the transforming DNA. The transformants' Ddc RNA is indistinguishable from that of wild type. One reintegrated Ddc gene, inserted on the X chromosome, is affected by the dosage compensation mechanism that leads to sex-specific differences in the expression of many X-chromosome genes.

A genetic analysis of tetrahymena that have aborted normal development.

Conjugating Tetrahymena can abort the sexual cycle before the production of progeny somatic (macro-) nuclei and return to vegetative growth. We have analyzed the germinal (micronuclear) genotypes of these cells in order to determine the stage at which they aborted conjugation. Our data demonstrate that nearly all of these cells attempt meiosis, but that very few reach the successful completion of cross-fertilization. The resulting micronuclear genotypes suggest that either germinal chromosomes or entire nuclei are lost during an unsuccessful attempt at meiosis or cross-fertilization. We conclude that the decision to develop progeny macronuclei is made during meiosis and is dependent on the completion of some step necessary for successful cross-fertilization.