Ectopic expression of guanylyl cyclase C in CD34+ progenitor cells in peripheral blood.

PURPOSE: To examine the utility of guanylyl cyclase C (GC-C)-specific nested reverse transcriptase polymerase chain reaction (RT-PCR) to detect circulating tumor cells in patients with colorectal cancer. PATIENTS AND METHODS: Peripheral-blood mononuclear cells from 24 patients with Dukes' stage D colorectal cancer were analyzed by GC-C-specific nested RT-PCR using 1 microg of total RNA. Peripheral-blood mononuclear cells from 20 healthy volunteers served as controls. Additionally, peripheral-blood CD34+ progenitor cells were assayed for the expression of both GC-C and other epithelial cell-specific markers. RESULTS: GC-C mRNA was detected in blood mononuclear cells from all 24 patients with colorectal cancer and all healthy volunteers. These unexpected positive results reflected low-level ectopic transcription of GC-C in CD34+ progenitor cells. Moreover, CD34+ progenitor cells expressed other epithelial cell-specific markers, including prostate-specific antigen, prostate-specific membrane antigen, carcinoembryonic antigen, CK-19, CK-20, mucin 1, and GA733.2. Limiting the quantity of mononuclear cell total RNA analyzed to < or = 0.8 microg eliminated detection of GC-C and other tissue-specific transcripts in blood of healthy volunteers. However, under the same conditions, GC-C mRNA was detected in mononuclear cells from all 24 patients with metastatic colorectal cancer. Using 0.5 microg of total RNA and GC-C-specific primers, nested RT-PCR detected a single human colon carcinoma cell (approximately 20 to 200 GC-C transcripts/cell) in 10(6) to 10(7) mononuclear blood cells. CONCLUSION: These data suggest that GC-C may be useful for detecting circulating colorectal cancer cells. They also demonstrate that CD34+ cells are a source of ectopically expressed epithelial cell-specific markers and that CD34+ cells may contribute to the high false-positive rate generally observed when those markers are used to detect rare circulating metastatic cancer cells by RT-PCR.

Postoperative management of local colorectal cancer: therapy and surveillance.

Adjuvant therapy is widely recommended for stage III colon cancer and stages II and III rectal cancer. Although fluorouracil-based regimens are standard, newer agents either alone or in combination may improve response rates. Although nearly all patients enter a postoperative surveillance program after surgical resection, the clinical effectiveness of such surveillance, which is not standardized, is questionable. Critical review of the use of different components (laboratory, radiographic, and endoscopic) of these programs finds little support for intensive surveillance.

Colorectal cancer staging and adjuvant chemotherapy.

Colorectal cancer is a significant cause of morbidity and mortality in Western populations. The standard of care for staging patients with colorectal cancer to determine prognosis and identify patients who will receive adjuvant therapy continues to be histopathology of regional lymph nodes. However, the significant variability in survival within each staging category likely reflects the heterogeneity of detecting micrometastatic disease employing this technique. Novel molecular markers of micrometastases currently in development will permit more accurate staging of patients with colorectal cancer. These advances in staging will distinguish patients who will maximally benefit from adjuvant therapy from those who have an especially good prognosis in whom chemotherapy can be avoided. In addition, new adjuvant chemotherapeutic agents, novel combinations of those agents and creative dosing schedules currently being investigated will offer considerable advantages with respect to ease of administration, safety and tolerability, quality of life and efficacy. Ultimately, it is anticipated that advances in molecular diagnostics will define unique biochemical characteristics of patients' tumours, permitting individualization of chemotherapeutic regimens employing novel agents that specifically exploit those characteristics.

Flow cytometric analysis and confocal imaging of anticancer alkylaminoanthraquinones and their N-oxides in intact human cells using 647-nm krypton laser excitation.

Flow cytometry and laser-scanning confocal fluorescence microscopy have been used in the study of the pharmacodynamics, in single intact cells, of two novel alkylaminoanthraquinones (AQ4 and AQ6), structurally based upon the mid-red excitable but very weakly fluorescent anticancer agent mitoxantrone, together with their respective N-oxide derivatives (AQ4NO and AQ6NO). The drug design rationale was that N-oxide modifications generates prodrug forms suitable for selective bioreductive-activation in hypoxic tumor cells. DNA-binding ranked in the order of mitoxantrone > AQ6 > AQ4 > AQ6NO >> AQ4NO. Using both cytometric methods a similar ranking was found for whole cell and nuclear location in human transformed fibroblasts. However, AQ6 showed enhanced nuclear uptake compared with mitoxantrone, in keeping with its greater capacity to inhibit DNA synthesis. Partial charge neutralisation by N-oxide derivatization resulted in loss of DNA synthesis inhibition but retention of the ability to accumulate in the cytosol, an important property for prodrug development. We conclude that both flow cytometry and confocal imaging revealed biologically significant differences between analogues for subcellular distribution and retention properties. The study demonstrates the potential for these complementary 647-nm krypton laser line-based fluorometric methods to provide relevant structure-activity information in anthraquinone drug-design programmes.

DNA topoisomerase II-dependent cytotoxicity of alkylaminoanthraquinones and their N-oxides.

We studied the role of DNA topoisomerase II in the biological actions of a series of novel alkylaminoanthraquinones, including N-oxide derivatives designed as prodrugs liable to bioreductive activation in hypoxic tumour cells. Drug structures were based upon the DNA-binding anticancer topoisomerase II poison mitoxantrone with modifications to the alkylamino side chains. The agents included AQ4, 1,4-bis{[2-(dimethylamino)ethyl] amino}5,8-dihydroxy-anthracene-9,10-dione, and AQ6, 1{[2-dimethylamino)-ethyl]amino}4-{[2[(hydroxyethyl)amino]ethyl]- amino}5,8-dihydroxy-anthracene-9,10-dione, together with the corresponding mono-N-oxide (AQ6NO) and di-N-oxide (AQ4NO). The R3N(+)-O- modification renders the terminal nitrogen group electrically neutral and was found to reduce AQ6NO or effectively abolish AQ4NO-DNA binding. Comparative studies were carried out using two SV40-transformed fibroblast cell lines, MRC5-V1 and AT5BIVA, the latter being a relative overproducer of DNA topoisomerase II alpha. The inhibition of DNA topoisomerase II decatenation activity ranked according to DNA-binding capacity. A similar ranking was found for drug-induced DNA-protein cross-linking in intact cells, depending upon topoisomerase II availability. Inhibition of DNA synthesis in S-phase synchronized cultures ranked in the order of AQ6 > mitoxantrone > > AQ6NO and was independent of topoisomerase II availability. Cytotoxicity of acute 1-h exposures for all agents except the inactive AQ4NO was enhanced in the topoisomerase II-overproducing cell line. The results indicate an important role for enzyme targeting in anthraquinone action. However, DNA synthesis inhibition and cytotoxicity were greater than expected for AQ6, given its topoisomerase- and DNA-interaction properties, and parallel studies have provided evidence of an additional role for enhanced subcellular accumulation and nuclear targeting. The inactivity of AQ4NO and the retention of only partial activity of AQ6NO, allied with the effective topoisomerase II-targeting and high cytotoxic potential of their presumed metabolites, favour their use as prodrugs in tumour cells with enhanced bioreductive potential.

First metatarsocuneiform joint arthrodesis: a five-year retrospective analysis.

Historically, first metatarsocuneiform joint arthrodesis has been advocated for severe metatarsus primus adductus deformity and as a salvage procedure in reconstructive forefoot surgery. At the Podiatry Hospital of Pittsburgh, the authors have expanded the use of this procedure to include elimination of excessive motion at the first metatarsocuneiform joint and to restore functional integrity to the first ray in an otherwise poorly functioning foot. First metatarsocuneiform arthrodesis has had poor acceptance by podiatric surgeons. Common reasons for this poor acceptance are technical difficulty associated with the procedure and often-cited complications such as shortening of the first ray, with symptomatic transfer lesions, as well as nonunion of the arthrodesis site. The authors evaluated 54 procedures. Results of first metatarsocuneiform joint arthrodesis for long-term reduction of the intermetatarsal angle and for limiting abnormal motion at this joint have been very good. Few complications were encountered. Shortening of the first metatarsal with secondary transfer lesions was not found to be a common problem. Several radiographic nonunions occurred, none of which were symptomatic. The surgical procedure, as it is performed at the Podiatry Hospital of Pittsburgh, is described. Results from a 5-year retrospective analysis of first metatarsocuneiform arthrodesis are presented.