Allelic loss on chromosome band 18p11.3 occurs early and reveals heterogeneity in breast cancer progression.

We examined the stage specificity and heterogeneity of 18p11 alterations in a series of tumors representing 96 microdissected samples. Significant loss of heterozygosity (LOH) (63%) was found, with 56% occurring early in ductal carcinoma in situ. Although most cases indicated LOH was clonally inherited, heterogeneity for 18p LOH occurred in 27% of tumors. When compared with other LOH data, 18p LOH was found in conjunction with allelic deletion on 3p, 9p, 17p and 17q, while 13q, 16q, and 11p were less frequently associated. These analyses suggest chromosome 18p11 alteration is a common and early event in breast disease.

Loss of DAL-1, a protein 4.1-related tumor suppressor, is an important early event in the pathogenesis of meningiomas.

Meningiomas are common nervous system tumors, whose molecular pathogenesis is poorly understood. To date, the most frequent genetic alteration detected in these tumors is loss of heterozygosity (LOH) on chromosome 22q. This finding led to the identification of the neurofibromatosis 2 (NF2) tumor suppressor gene on 22q12, which is inactivated in 40% of sporadic meningiomas. The NF2 gene product, merlin (or schwannomin), is a member of the protein 4.1 family of membrane-associated proteins, which also includes ezrin, radixin and moesin. Recently, we identified another protein 4.1 gene, DAL-1 (differentially expressed in adenocarcinoma of the lung) located on chromosome 18p11.3, which is lost in approximately 60% of non-small cell lung carcinomas, and exhibits growth-suppressing properties in lung cancer cell lines. Given the homology between DAL-1 and NF2 and the identification of significant LOH in the region of DAL-1 in lung, breast and brain tumors, we investigated the possibility that loss of expression of DAL-1 was important for meningioma development. In this report, we demonstrate DAL-1 loss in 60% of sporadic meningiomas using LOH, RT-PCR, western blot and immunohistochemistry analyses. Analogous to merlin, we show that DAL-1 loss is an early event in meningioma tumorigenesis, suggesting that these two protein 4.1 family members are critical growth regulators in the pathogenesis of meningiomas. Furthermore, our work supports the emerging notion that membrane-associated alterations are important in the early stages of neoplastic transformation and the study of such alterations may elucidate the mechanism of tumorigenesis shared by other tumor types.

An acute, traumatic supraspinatus lesion in an intercollegiate football player: a case report.

OBJECTIVE: To describe the evaluation, management, and rehabilitation of an acute, supraspinatus tendon injury in an intercollegiate football player. BACKGROUND: While attempting to block a defender, a 19-year-old collegiate football player slipped on the artificial turf and landed on his right elbow, causing an injury to his right shoulder. The athlete was initially seen by the head athletic trainer and then referred to the team physician for further evaluation. DIFFERENTIAL DIAGNOSIS: acromioclavicular joint sprain, brachial plexopathy, subacromial impingement syndrome, supraspinatus lesion. TREATMENT: The athlete was managed surgically with an open acromioplasty and a 3-bone tunnel repair of the supraspinatus tendon. After surgery, the athlete underwent a 4-month rehabilitation protocol in preparation for return to competition. UNIQUENESS: This case involved a teenage athlete rather than the older individuals who normally sustain rotator cuff lesions. Also, the mechanism was a compressive force on the supraspinatus tendon rather than the tensile force common to rotator cuff lesions. CONCLUSIONS: By presenting this case report, we hope to give sports medicine clinicians a better understanding of rotator cuff injuries and how to successfully manage and rehabilitate supraspinatus lesions.

Use of horizontal ultrathin gel electrophoresis to analyze allelic deletions in chromosome band 11p15.5 in gliomas.

The prognosis for most patients with astrocytic glioma is poor, and postoperative life expectancy has not significantly improved in the last decade despite advances in diagnosis, surgery, and adjuvant therapy. Progress has been made, however, in cataloging the genetic alterations that occur in these tumors. Studying the allelic changes using loss of heterozygosity analysis has proven to be a reliable and rapid way of identifying genetic alterations fundamental to the pathology of this disease. In this study, we used a series of fluorescent-labeled markers and a new horizontal ultrathin gel electrophoresis technology (HUGE; GeneSys Technologies, Inc.) to analyze loss of heterozygosity on 11p15 in a series of 24 matched normal/tumor glioma pairs that included both anaplastic astrocytomas and glioblastomas. These studies significantly narrowed the region harboring a putative 11p15.5 glioma-associated gene and further suggest that a second gene involved in the pathogenesis of brain tumors may exist, centromeric, in bands 11p15.5-p15.4.

A novel member of the NF2/ERM/4.1 superfamily with growth suppressing properties in lung cancer.

A novel putative tumor suppressor gene and member of the NF2/ERM/ 4.1 superfamily was isolated using Differential Display PCR (DDPCR) on primary lung tumors. When reintroduced into nonexpressing non-small cell lung carcinoma cell lines, this gene, named DAL-1 (for Differentially expressed in Adenocarcinoma of the Lung), was shown to suppress growth. In addition, significantly reduced expression (>50%) of DAL-1 was measured in 39 primary non-small cell lung carcinoma tumors as compared with patient-matched normal lung tissue. Immunocytochemical staining with a polyclonal anti-DAL-1 antibody localized the protein to the plasma membrane, particularly at cell-cell contact points, a pattern reminiscent of other members of the protein 4.1 superfamily including ezrin and NF2. The data suggest DAL-1 is a novel membrane-associated protein with potential to play an important role in the origin and progression of lung cancer.

Novel regions of allelic deletion on chromosome 18p in tumors of the lung, brain and breast.

Lung cancer is now the number one cause of cancer death for both men and women. An age-adjusted analysis over the past 25 years shows that in women specifically, lung cancer incidence is on the rise. It is estimated that 10-20 genetic events including the alteration of oncogenes and tumor suppressor genes will have occurred by the time a lung tumor becomes clinically evident. In an effort to identify regions containing novel cancer genes, chromosome 18p11, a band not previously implicated in disease, was examined for loss of heterozygosity (LOH). In this study, 50 matched normal and NSCLC tumor samples were examined using six 18p11 and one 18q12.3 PCR-based polymorphic markers. In addition, LOH was examined in 29 glioblastoma pairs and 14 paired breast carcinomas. This analysis has revealed potentially two regions of LOH in 18p11 in up to 38% of the tumor samples examined. The regions of LOH identified included a 2 cm area between markers D18S59 and D18S476, and a more proximal, 25 cm region of intermediate frequency between D18S452 and D18S453. These results provide evidence for the presence of one or more potential tumor suppressor genes on the short arm of chromosome 18 which may be involved in NSCLC, brain tumors and possibly breast carcinomas as well.

Conditioning injuries associated with artificial turf in two preseason football training programs.

OBJECTIVE: To compare the occurrence of preseason football conditioning injuries in traditional and crossover conditioning programs over five preseasons. DESIGN AND SETTING: The turf group performed all preseason conditioning by running or sprinting on artificial turf, and the turf and swim group alternated all preseason conditioning by running and sprinting on artificial turf or kickboard swimming. SUBJECTS: Subjects were 519 NCAA Division III physically active football players. MEASUREMENTS: Previous conditioning injuries, preseason conditioning injuries, missed practices, and missed conditioning sessions were recorded. Exact numbers and areas of injury for each year and each group were tabulated. A chi-square statistic compared the two groups and a logistic regression model was used to estimate the risk of becoming injured and the types of conditioning injuries experienced in the two groups. RESULTS: Prevalence of injury was significantly different in the two groups. In the turf group, 35% of subjects developed a conditioning injury; in the turf and swim group, 13% developed a conditioning injury (x(2)= 33.16, p <.0001). No significant difference in missed practices or missed conditioning sessions was found. CONCLUSIONS: The turf and swim group experienced significantly fewer football preseason conditioning injuries than the turf group. The crossover effects of a running and swimming program may decrease the number of overuse injuries associated with repetitive running on artificial turf in traditional preseason conditioning programs.

Characterization of a rearrangement in the c-MYB promoter in the acute lymphoblastic leukemia cell line CCRF-CEM.

Despite the frequent description of 6q- structural abnormalities in human leukemias and lymphomas, rearrangements of the c-MYB locus have not been detected. We have detected a rearrangement in the c-MYB proto-oncogene in the cell line CCRF-CEM, an immature T-cell leukemia cell line which is not 6q-. Due to this rearrangement, a large portion of the c-MYB promoter conserved between the human and murine c-MYB genes is lost. The rearranged locus, which we have designated MRR (MYB rearranged region), has been cloned and mapped to chromosome 6. Field inversion gel electrophoresis (FIGE) studies reveal that the MRR sequence is linked to the c-MYB locus, suggesting that the rearrangement is due to a submicroscopic deletion. The rearrangement appears to have no effect on c-MYB promoter activity as analyzed in CCRF-CEM cells. The normal locus of the MRR sequence has been cloned from a human placental genomic library. Partial sequence analysis of this clone reveals that a portion of the DNA lost in the rearrangement shows a high degree of homology to a member of the myc family of oncogenes. Thus the characterization of this rearrangement has yielded a new set of probes for the study of chromosome 6q abnormalities in human leukemias and lymphomas and provides the first evidence for potential involvement of the c-MYB locus itself in submicroscopic deletions within chromosome 6.

Identification of a second promoter in the human c-myb proto-oncogene.

We have previously described an alternatively spliced cDNA clone of the human c-myb proto-oncogene which has been shown to enhance the differentiation of Friend murine erythroleukemia cells. This clone, pMbm-2, contains unique 5' sequences which replace exon 1. The human c-myb intron 1 was sequenced to determine the exact position of this unique sequence and to further characterize the role of intron 1 in the regulation of the human c-myb gene. Here we report that intron 1 of c-myb is highly conserved between human and mouse throughout the intron, while only those sequences directly adjacent to exons 1 and 2 are conserved between human and chicken. The unique sequence of pMbm-2 was located directly adjacent to exon 2, suggesting that it arose as a product of alternative transcription initiation within intron 1. RNAase protection analysis was used to map a cluster of transcription start sites at the 5' end of exon 2. Levels of messages utilizing these start sites are proportional to those arising from the primary promoter. Functional characterization of this region revealed that this region can function as a promoter. Deletion studies have revealed the presence of negative and positive regulatory elements within this region which are utilized with different efficiencies in different cell lines. These studies suggest that cis or trans factors acting in this region may serve a dual function in both attenuation and transcription initiation.

Multiple mechanisms of regulation of the human c-myb gene during myelomonocytic differentiation.

Alterations in expression of c-myb can have profound effects on the growth and differentiation of hematopoietic cells. Thus, it is important to understand the mechanisms by which c-myb is regulated during hematopoietic cell differentiation. Previous studies pertaining to the regulation of c-myb have been carried out with the avian and murine forms of the gene; the current studies were designed to determine the mechanisms of regulation of the human form of c-myb. Transcriptional analysis by nuclear run-on assays revealed that an attenuation of transcription was the means of primary regulation during retinoic acid- and vitamin D3-induced differentiation of HL-60 cells, while other mechanisms in addition to attenuation were active during dimethyl sulfoxide (DMSO)- and phorbol ester-induced differentiation. Densitometric analysis of the changes in c-myb transcription caused by phorbol ester suggested that the c-myb promoter may be down-regulated during phorbol ester-induced differentiation of HL-60. Additional studies exhibited post-transcriptional regulation by phorbol ester. DMSO was also shown to regulate c-myb at the post-transcriptional level. Interestingly, the post-transcriptional regulation of c-myb by DMSO required continuous transcription. This requirement was shared for c-myc but not ornithine decarboxylase expression. The transcriptional dependency of c-myb post-transcriptional regulation did not equate to translational dependency, thus a novel post-transcriptional regulatory mechanism may control c-myb gene expression. The multiple levels of regulation of c-myb suggest the importance of proper control for hematopoietic cell differentiation.

Alternative splicing of the human c-myb gene.

Two cDNA clones of the human c-myb gene have been isolated from a CCRF-CEM leukemia cell cDNA library and sequenced in their entirety. These sequences, when compared with those previously reported for the human c-myb gene, reveal an alternative splicing process that generates at least four forms of the c-myb message. Three of these forms co-migrate on Northern blots and are co-expressed in several human hematopoietic cell types. Data on sequence comparisons with mouse and chicken homologues of c-myb coupled with oligonucleotide hybridization to genomic clones of the human c-myb gene indicate that this alternative splicing process utilizes three closely spaced splice donor sites and two unique exons present between viral defined exons 5 and 6. In one clone, the alternative splicing would generate a predicted myb protein with a three amino acid deletion in the region involved in transcription activation. In the other clone, incorporation of a new exon leads to introduction of a translation stop codon leading to loss of the entire carboxy terminus of the protein. This includes loss of a portion of the region involved in transcription activation as well as a separate highly conserved domain. The effect of these changes on protein function is currently unknown.

Transient inhibition of DNA synthesis by 5-fluorodeoxyuridine leads to overexpression of dihydrofolate reductase with increased frequency of methotrexate resistance.

Transient but incomplete suppression of DNA synthesis by a single exposure of an asynchronous population of cells to 5-fluoro-2'-deoxyuridine (FdUrd) increases the frequency of appearance of methotrexate (MTX)-resistant colonies. This increase was greater than 10-fold following a 6-h incubation of cells with 3 microM FdUrd prior to selection in MTX, an interval one-half the normal L1210 cell cycle time. During this period of exposure to FdUrd, DNA synthesis decreased to 25% of control rates and cells accumulated at the G1/S interface. The 6-h incubation with FdUrd resulted in greater than a 2.5-fold increase in the dihydrofolate reductase protein level in the treated cell population, which was accounted for, at least in part, by increased de novo synthesis of the enzyme as assessed by [35S]methionine labeling. This increase in dihydrofolate reductase was associated with a decrease in growth inhibition by MTX. A brief reversal (2 h) of FdUrd-induced DNA synthesis inhibition by the addition of thymidine eliminated the amplification of dihydrofolate reductase and the enhanced emergence of MTX-resistant clones. Beyond this, an analysis of clones that survive MTX selection indicates that the dihydrofolate reductase gene copy in cells spontaneously resistant to 50 nM MTX and those which resulted after the additional pretreatment with FdUrd for 6 h are comparable with a 2-4-fold amplification of enzyme in most clones. These studies demonstrate that FdUrd enhancement of dihydrofolate reductase expression can have a profound effect upon the incidence and expression of MTX resistance and that dihydrofolate reductase gene amplification may be another basis for antagonism between these agents.