Wild-type p53 and p73 negatively regulate expression of proliferation related genes.

When normal cells come under stress, the wild-type (WT) p53 level increases resulting in the regulation of gene expression responsible for growth arrest or apoptosis. Here we show that elevated levels of WT p53 or its homologue, p73, inhibit expression of a number of cell cycle regulatory and growth promoting genes. Our analysis also identified a group of genes whose expression is differentially regulated by WT p53 and p73. We have infected p53-null H1299 human lung carcinoma cells with recombinant adenoviruses expressing WT p53, p73 or beta-galactosidase, and have undertaken microarray hybridization analyses to identify genes whose expression profile is altered by p53 or p73. Quantitative real-time PCR verified the repression of E2F-5, centromere protein A and E, minichromosome maintenance proteins (MCM)-2, -3, -5, -6 and -7 and human CDC25B after p53 expression. 5-Fluorouracil treatment of colon carcinoma HCT116 cells expressing WT p53 results in a reduction of the cyclin B2 protein level suggesting that DNA damage may indeed cause repression of these genes. Transient transcriptional assays verified that WT p53 repressed promoters of a number of these genes. Interestingly, a gain-of-function p53 mutant instead upregulated a number of these promoters in transient transfection. Using promoter deletion mutants of MCM-7 we have found that WT p53-mediated repression needs a minimal promoter that contains a single E2F site and surrounding sequences. However, a single E2F site cannot be significantly repressed by WT p53. Many of the genes identified are also repressed by p21. Thus, our work shows that WT p53 and p73 repress a number of growth-related genes and that in many instances this repression may be through the induction of p21.

Matching resources to care: the acceptability, validity and inter-rater reliability of a new instrument to assess severe mental illness (MARC-1).

BACKGROUND: Most definitions of severe mental illness (SMI) are categorical and assign the patient to either SMI or not-SMI status. While this is useful for some purposes, it is a rather limited approach. The purpose of the present study is to develop a new method of addressing the issue of 'severity', and to develop a dimensional rather than a categorical approach. The paper reports on the acceptability, reliability and validity of a method developed to collect a standard set of data covering the majority of items specified in the academic and policy literature as characterising SMI. METHOD: A single page form, Matching Resources to Care (MARC-1), containing most of the items used in definitions of SMI was used to collect data from community mental health staff about their current open caseload, in four co-terminous health and social services settings during a census week (n = 2139). In addition to the data from the four pilot sites, we conducted a substudy (n = 91), in which two raters rated the same cases during the same week. RESULTS: The MARC-1 scores were able to distinguish between patients in receipt, and those not in receipt, of specific types of community care (level of care, eligibility for care and statutory aftercare) (P < 0.001). The MARC-1 score was modestly but significantly correlated (r = 0.28) with the Global Assessment Scale (P < 0.001). The mean percentage inter-rater agreement for the MARC-1 score items was 87%. CONCLUSION: It is possible to use a simple census form in both health and social services agencies. The completion rates were good in both services. The levels of reliability were good, and concurrent validity was established with specific types of care in the community.

Effect of dGTP concentration on human and CHO telomerase.

Human telomerase produces a long ladder of six-base repeat additions to a primer, while CHO telomerase primarily adds only one or two repeat additions to a primer. Under the standard assay conditions, the concentration of dGTP is very low, so we investigated the effects of increasing dGTP concentration on human and CHO telomerase activities. Increasing dGTP concentration over a range of 1.5-50 microM caused the human telomerase to produce longer primer extension products until products were so large that no ladder pattern was apparent. Increasing dGTP concentration resulted in CHO telomerase producing one to eight repeat additions, though still not as many repeats as produced by human telomerase even under low dGTP conditions. CHO telomerase produced a six-base ladder pattern comparable to human telomerase only after raising the dGTP concentration to 500 microM under conditions in which the dATP concentration was low. Primer challenge experiments showed the human telomerase exhibited approximately 100% processivity at both low and high concentrations of dGTP, and thus increasing dGTP concentration appeared to affect only the extension rate. In contrast, CHO telomerase exhibited low processivity under low concentrations of dGTP and increased processivity at higher dGTP concentrations. One explanation for the low processivity of CHO was found in CHO telomerase's inability to extend the GGTTAG permuted primer under nonprocessive conditions, while able to extend the other five permuted primers. Competition studies of different permuted primers indicated that the GGTTAG primer cannot interact with the nonprocessive CHO telomerase. A model is proposed for explaining the nonprocessive behavior of CHO telomerase.

A human breast cancer model for the study of telomerase inhibitors based on a new biotinylated-primer extension assay.

Telomerase is an RNA-dependent polymerase that synthesizes telomeric DNA (TTAGGG)n repeats. The overall goal of our work was to establish human cancer models that can be used to design clinical trials with telomerase inhibitors. The objectives of this study were (1) to set up a human breast cancer system that allows evaluation of the effects of telomerase inhibitors in cultured cells using a non-amplified telomerase assay and (2) to test this system using two drugs (cisplatin and TMPyP4) that affect the telomerase expression in breast cancer cells in culture. We first compared the telomerase activity in a variety of human breast cancer cell lines to that of other tumour types using a new biotinylated-primer extension assay. Our method, based on a non-amplified primer extension assay shows the direct incorporation of 32P-labelled nucleotides induced by telomerase on human telomeric primers. The 32P-dGTP labelled telomerase-extended 5'-biotinylated (TTAGGG)3 primer can subsequently be separated using streptavidin-coated magnetic beads. As compared to other non-amplified method, we showed that this procedure improved the characterization and the quantification of the banding pattern resulting from telomerase extension by reducing the radioactive background. Using this method, we observed that telomerase activity varies markedly in a panel of 39 human cancer cell lines. For example, MCF7 breast cancer cells in culture showed intermediate telomerase activity corresponding to 33.8+/-3.4% of that of the HeLa cells (reference cell line). Similarly, the telomere length varied with each cell line (average: 6.24+/-6.16). No correlation between the level of telomerase and telomere length was observed, suggesting that a high processivity is not required to maintain telomeres and that, in some cell lines, another mechanism of telomere elongation can maintain telomere length. From this study, we selected MCF7 and MX1 models that showed reproducible telomerase activity and a relatively limited telomere length for the testing of potential telomere-telomerase interacting agents. Using cisplatin and a new porphyrin-derived compound TMPyP4, we showed that our model was able to detect a down-regulation of the telomerase activity in MCF7 cells in culture and in a human MX1 tumour xenografts. Based on these results, a breast cancer model for evaluating telomerase and telomere interactive agents is proposed.

Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells.

A series of cationic porphyrins has been identified as G-quadruplex interactive agents (QIAs) that stabilize telomeric G-quadruplex DNA and thereby inhibit human telomerase; 50% inhibition of telomerase activity was achieved in HeLa cell-free extract at porphyrin concentrations in the range < or = 50 microM. Cytotoxicity of the porphyrins in vitro was assessed in normal human cells (fibroblast and breast) and human tumor cells representing models selected for high telomerase activity and short telomeres (breast carcinoma, prostate, and lymphoma). In general, the cytotoxicity (EC50, effective concentration for 50% inhibition of cell proliferation) against normal and tumor cells was > 50 microM. The porphyrins were readily absorbed into tumor cell nuclei in culture. Inhibition of telomerase activity in MCF7 cells by subcytotoxic concentrations of TMPyP4 showed time and concentration dependence at 1-100 microM TMPyP4 over 15 days in culture (10 population doubling times). The inhibition of telomerase activity was paralleled by a cell growth arrest in G2-M. These results suggest that relevant biological effects of porphyrins can be achieved at concentrations that do not have general cytotoxic effects on cells. Moreover, the data support the concept that a rational, structure-based approach is possible to design novel telomere-interactive agents with application to a selective and specific anticancer therapy.

AZT induces high frequency, rapid amplification of centromeric DNA.

The reverse transcriptase inhibitor 3'-azido-deoxythymidine (AZT) has previously been shown to be incorporated into specific regions near the telomeres and centromeres of Chinese hamster ovary cell chromosomes. Our investigation of the effects of AZT on chromosome stability has led to the discovery of a high frequency amplification of telomere-like centromeric DNA. The amplified structures, when analyzed cytogenetically, appear as tandem arrays of tightly clustered blocks of centromeric repeats containing telomeric sequences (TTAGGG)n. There were 5-13 blocks of amplified DNA per structure. These structures form rapidly within one or two cell cycles and can be observed with an incidence as high as 2%. Because the amplification was so rapid, we tested whether the amplification structures could be the result of aberrant overreplication by analyzing BrdU incorporation. Our results indicate that the amplified DNA does not undergo abnormal replication during its formation, but appears to form from existing centromeric regions. We propose a model that involves the excision of multiple centromeric DNA regions from other chromosomes and their relocalization to a new site.

Preclinical and clinical strategies for development of telomerase and telomere inhibitors.

BACKGROUND: Telomerase is an important enzyme whose activity has been convincingly demonstrated in humans recently. It is required for maintenance of ends of chromosomes (telomeres) during cell division. Since its presence has been selectively demonstrated in dividing cells including tumor cells, it has generated considerable excitement as a potential anti-cancer strategy. DESIGN: In this article, we review the current relevant biology of the enzyme, the challenges encountered in the preclinical phase of target development and the current efforts that focus on telomeres and telomerase as therapeutic targets. We also speculate on the potential toxicities and mechanisms of resistance that may be encountered during use of such therapies.

Agents that target telomerase and telomeres.

Telomeres are guanine-rich regions that are located at the ends of chromosomes and are essential for preventing aberrant recombination and protecting against exonucleolytic DNA degradation. Telomeres are maintained by telomerase, an RNA-dependent DNA polymerase. Because telomerase is known to be expressed in tumor cells, which concurrently have short telomeres, and not in most somatic cells, which usually have long telomeres, telomerase and telomere structures have been recently proposed as attractive targets for the discovery of new anticancer agents. The most exciting current strategies are aimed at specifically designing new drugs that target telomerase or telomeres and new models have been formulated to study the biological effects of inhibitors of telomerase and telomeres both in vitro and in vivo.

Basal cell carcinoma in the groin of a black patient with femoral vessel invasion and a second primary malignancy.

We have presented an unusual case of basal cell carcinoma that presented in a 76-year-old black woman. This is an atypical case in that it occurred in a black patient, and the incidence of basal cell carcinoma is low in black people. The lesion was located in the groin, which is an uncommon location for basal cell carcinoma. The femoral vessels in this patient also were invaded by the tumor, which is also a very rare occurrence. The patient in this report had a coexisting squamous cell carcinoma of the lung, which may be a common finding in black patients with basal cell carcinoma. It is extremely important for the physician to include skin cancer in the differential diagnosis of any suspicious skin lesion. This will avoid delays in treatment and decrease the overall morbidity. The physician who encounters a black patient with a basal cell carcinoma also should screen the patient for a coexisting noncutaneous malignancy.

The inferiorly based rectus abdominis island flap for the treatment of complex hip wounds.

Complex wounds in the hip region often result from complications of orthopedic procedures performed in this region such as total hip arthroplasty, ORIF procedures, tumor ablation, and/or radiation therapy. Exposure of bone, joint capsule, prostheses, and other hardware often results with these wounds. Salvage of these exposed and/or infected essential elements and providing soft-tissue coverage are difficult and challenging problems for the orthopedic and plastic surgeon. To provide coverage for such situations, we have developed an aggressive strategy of thorough debridement, systemic antibiotics, and well-vascularized soft-tissue coverage utilizing an inferiorly based rectus abdominis island flap. This technique was utilized in five patients with all wounds and joints remaining stable at follow-up periods ranging from 2 to 7 years.

A new suction device for microsurgery.

A new suction device for microsurgical procedures is presented. This device is made of soft, compliant PVC, which enables all fluids to be evacuated from the surgical field without damaging the surrounding soft tissue. The device is designed with front and radial ports that allow placement onto the blood vessel or nerve while the anastomosis is being performed. There is a tapered end providing the surgeon with fine control of the suction. Unlike other bulky suction devices, this one readily fits into the operative field without inhibiting operative procedures. It also readily fits varying sizes of conventional Frazier-type tips. The device has been used in over 100 microsurgical procedures and it has served well.

A new vessel occluder for microvascular anastomoses.

A vessel occluder has been developed that is easy to use and provides effective, safe, vascular occlusion. The occluder is small and is easily rotatable in the operative field. It consists of a plastic backing and a compliant foam strap that wraps around the blood vessel. The foam absorbs blood and irrigation which keeps the vessels moist during the anastomosis. This vessel occluder possesses the ability to vary the amount of blood flow through the vessel, enabling the surgeon to identify and readily repair anastomotic leaks. This occluder has been used successfully by the authors in 100 anastomoses.

Anatomical parameters for nipple position and areolar diameter in males.

In this paper we present anatomic parameters for nipple position and areolar diameter in males. Larger forms of gynecomastia with significant ptosis pose a challenge to the plastic surgeon with respect to relocation of the nipples on the chest wall. Selection of the appropriate areolar size is also of concern in gynecomastia correction. There is a paucity of information in the current literature pertaining to this problem. In order to establish guidelines for the placement of the nipple in gynecomastia correction and for the selection of the appropriate areolar size, we set out to determine these anatomic parameters. We believe use of these parameters will enhance the aesthetic results of gynecomastia correction. One hundred males between the ages of 17 to 30 years were chosen for this study. The males selected were of ideal body weight and without evidence of gynecomastia. The distances from the sternal notch to the nipple, the midclavicular line to the nipple, and the nipple-to-nipple distance were recorded. The areolar diameter was also measured in each subject. The average distances were determined for each category. The validity of these values was confirmed with statistical analysis. Equations were then derived, using this analysis, to determine nipple position in males. We have determined the nipple position in males to be approximately 20 cm from the sternal notch and 18 cm from the midclavicular line. The ideal nipple-to-nipple distance is 21 cm. The average areolar diameter is 2.8 cm.

Basal cell carcinoma in black patients: the need to include it in the differential diagnosis.

We present a retrospective study of 276 basal cell carcinomas which we have identified 5 (1.8%) black patients. This finding agrees with the current literature, which states that basal cell carcinoma in the black population is relatively infrequent. Although basal cell carcinoma in black patients is uncommon, it should be included in the differential diagnosis of any suspicious lesion in this population to avoid the morbidity that is associated with a delay in diagnosis. In this study we also present the possible association of basal cell carcinoma occurring concomitantly with a second primary malignancy in this population. In black patients basal cell carcinoma is found more frequently in regions of the body that are protected from ultraviolet radiation when compared to white patients. This leads to the speculation that a different pathogenesis of basal cell carcinoma exists for black patients.

Supplemental oxygen: ensuring its safe delivery during facial surgery.

Electrosurgical coagulation in the presence of blow-by oxygen is a potential source of fire in facial surgery. A case report of a patient sustaining partial-thickness facial burns secondary to such a flash fire is presented. A fiberglass facial model is then used to study the variables involved in providing supplemental oxygen when an electrosurgical unit is employed. Oxygen flow, oxygen delivery systems, distance from the oxygen source, and coagulation current levels were varied. A nasal cannula and an adapted suction tubing provided the oxygen delivery systems on the model. Both the "displaced" nasal cannula and the adapted suction tubing ignited at a minimum coagulation level of 30 W, an oxygen flow of 2 liters/minute, and a linear distance of 5 cm from the oxygen source. The properly placed nasal cannula did not ignite at any combination of oxygen flow, coagulation current level, or distance from the oxygen source. Facial cutaneous surgery in patients provided supplemental oxygen should be practiced with caution when an electrosurgical unit is used for coagulation. The oxygen delivery systems adapted for use are hazardous and should not be used until their safety has been demonstrated.

High resolution microscopic mapping of DNA using multi-color fluorescent hybridization.

We describe a procedure for microscopically mapping the relative positions of DNA probes along extended strands of DNA. The procedure referred to as direct visual hybridization (DIRVISH) DNA mapping involves the simultaneous hybridization of multiple probes and the fluorescent colors, red green and blue to produce images that convey high-resolution mapping information. The images appear as long strings of fluorescent signals positioned as they are in the genome. A visual multi-color map is generated within 2 days. Cosmid probes span a distance of 10 microms or more and have been observed to contain patterns within the strings of signals. We have developed computer imaging programs to scan through the strings of signals and plot the intensities. Scans through multiple signal strings for one cosmid probe revealed consistent patterns. We have interpreted the patterns as the result of suppression of repetitive DNA sequence hybridization. These patterns may prove useful as fingerprints for regions of DNA.

High resolution visual mapping of stretched DNA by fluorescent hybridization.

We describe a method for stretching DNA, which, when combined with fluorescent hybridization procedures, forms a new mapping technology that produces a high resolution, vivid, multi-colour image and map. Restriction fragments and cosmid probes were successfully mapped by this procedure with validation by standard restriction mapping. A long range map of a > 200 kilobase region containing five copies of the amplified dihydrofolate reductase gene was easily generated within two days. This DNA mapping procedure offers a significant and rapid alternative to a variety of standard mapping procedures.

c-myc oncogene copy number in squamous carcinoma of the head and neck.

PURPOSE: Altered resident cellular genetic sequences (oncogenes) may result in malignant transformation, maintenance of tumor growth, and metastatic propensity. In this pilot study, we have elected to probe c-myc oncogene in evaluating specimens from human squamous cell carcinoma. MATERIALS AND METHODS: Samples were obtained from 24 patients with squamous cell carcinoma of the head and neck. The ratio of tumor DNA values to that of control DNA was used to estimate the c-myc copy number. RESULTS: Data from material obtained from eight patients was analyzed to the point of c-myc copy number. Tumors varied from stage II through IV. Five originated in the oral cavity and three in the larynx. Analysis of primary tumors demonstrated that two of eight had increased c-myc copy numbers. Histologically positive neck specimens were encountered in five of the study patients. Three demonstrated elevated c-myc copy numbers, two of which had had increased copy number at the primary site. CONCLUSION: This study confirms that c-myc amplification can be present in squamous cell carcinoma of the head and neck. c-myc Amplification may also be present in neck metastasis. Oncogene amplification in neck metastasis may indicate an increased metastatic propensity for individual tumor cells demonstrating c-myc amplification.

A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

A CHO cell line with a single copy of the DHFR locus on chromosome Z2 was used to analyze the structure of the amplification target and products subsequent to the initial amplification event. Dramatic diversity in the number and cytogenetic characteristics of DHFR amplicons was observed as soon as eight to nine cell doublings following the initial event. Two amplicon classes were noted at this early time: Small extrachromosomal elements and closely spaced chromosomal amplicons were detected in 30-40% of metaphases in six of nine clones, whereas three of nine clones contained huge amplicons spanning greater than 50 megabases. In contrast, the incidence of metaphases containing extrachromosomal amplicons fell to 1-2% in cells analyzed at 30-35 cell doublings, and most amplicons localized to rearranged or broken derivatives of chromosome Z2 at this time. Breakage of the Z2 chromosome near the DHFR gene, and deletion of the DHFR gene and flanking DNA was also observed in cells that had undergone the amplification process. To account for these diverse cytogenetic and molecular consequences of gene amplification, we propose that chromosome breakage plays a central role in the amplification process by (1) generating intermediates that are initially acentric and lead to copy number increase primarily by unequal segregation, (2) creating atelomeric ends that are either incompletely replicated or resected by exonucleases to generate deletions, and (3) producing recombinogenic ends that provide preferred sites for amplicon relocalization.