Analogs of the ATP-Sensitive Potassium (K_ATP) Channel Opener Cromakalim with in Vivo Ocular Hypotensive Activity

ATP-sensitive potassium (K_ATP) channel openers have emerged as potential therapeutics for the treatment of glaucoma, lowering intraocular pressure (IOP) in animal models and cultured human anterior segments. We have prepared water-soluble phosphate and dipeptide derivatives of the K_ATP channel opener cromakalim and evaluated their IOP lowering capabilities in vivo. In general, the phosphate derivatives proved to be more chemically robust and efficacious at lowering IOP with once daily dosing in a normotensive mouse model. Two of these phosphate derivatives were further evaluated in a normotensive rabbit model, with a significant difference in activity observed. No toxic effects on cell structure or alterations in morphology of the aqueous humor outflow pathway were observed after treatment with the most efficacious compound, (3<i>S</i>,4<i>R</i>)-<b>2</b>, suggesting that it is a strong candidate for development as an ocular hypotensive agent

Clone gains/losses by ALT activity.

<p>Tumors that were ALT- exhibited significantly more chromosomal gains/losses than ALT+ rectal cancer (p=0.0091).</p

Histology, C-circle dot blot and aCGH summary for a MSS CIN- ALT + rectal cancer without activation of telomerase and MSS CIN+ ALT - rectal cancer with activation of telomerase.

<div><p>Panel A. Hematoxylin and Eosin tissue sections from an MSS CIN- , ALT+,Telomerase- rectal cancer (left) and from MSS CIN+, ALT-, Telomerase + rectal cancer. Both are moderately differentiated adenocarcinomas. The gland-to-stroma ratio is higher in the ALT+/tel- case, and it has less desmoplastic stroma.</p> <p>Panel B. Dot/blot showing presence of C-circles. C circles, extrachromosomal telomeric DNA, are strongly associated with ALT. Assessed in tumor DNA with isothermic amplification of C-circle complementary strand and hybridization with ³²P-(CCCTAA)₃ probe by Capital Biosciences (Capital Biosciences, Maryland, U. S. A. ), a sample was called ALT+ if C-circles were detected. The presence of C-circles are illustrated by the presence of radioactive tracer in the image on the left, and the absence of radioactivity in the blot on the right indicates absence of C-circles in the ALT- tumor. </p> <p>Panel C. Ideograms summarizing chromosomal gains and losses across all chromosomes evaluated by aCGH. The ALT+, telomerase negative tumor on the left had <10% of BAC clones showing aberrant hybridization and is classified as a CIN- tumor. The ALT-,,telomerase positive tumor on the right had 40% of clones with aberrant hybridization and is classified as a CIN+ tumor. </p> <p>Panel D. aCGH results of raw data for chromosome 17 for each tumor corresponding to the ideograms in Panel C. </p></div

Correlation of Chromosomal Instability, Telomere Length and Telomere Maintenance in Microsatellite Stable Rectal Cancer: A Molecular Subclass of Rectal Cancer

<div><p>Introduction</p><p>Colorectal cancer (CRC) tumor DNA is characterized by chromosomal damage termed chromosomal instability (CIN) and excessively shortened telomeres. Up to 80% of CRC is microsatellite stable (MSS) and is historically considered to be chromosomally unstable (CIN+). However, tumor phenotyping depicts some MSS CRC with little or no genetic changes, thus being chromosomally stable (CIN-). MSS CIN- tumors have not been assessed for telomere attrition. </p> <p>Experimental Design</p><p>MSS rectal cancers from patients ≤50 years old with Stage II (B2 or higher) or Stage III disease were assessed for CIN, telomere length and telomere maintenance mechanism (telomerase activation [TA]; alternative lengthening of telomeres [ALT]). Relative telomere length was measured by qPCR in somatic epithelial and cancer DNA. TA was measured with the TRAPeze assay, and tumors were evaluated for the presence of C-circles indicative of ALT. p53 mutation status was assessed in all available samples. DNA copy number changes were evaluated with Spectral Genomics aCGH. </p> <p>Results</p><p>Tumors were classified as chromosomally stable (CIN-) and chromosomally instable (CIN+) by degree of DNA copy number changes. CIN- tumors (35%; n=6) had fewer copy number changes (<17% of their clones with DNA copy number changes) than CIN+ tumors (65%; n=13) which had high levels of copy number changes in 20% to 49% of clones. Telomere lengths were longer in CIN- compared to CIN+ tumors (p=0.0066) and in those in which telomerase was not activated (p=0.004). Tumors exhibiting activation of telomerase had shorter tumor telomeres (p=0.0040); and tended to be CIN+ (p=0.0949).</p> <p>Conclusions</p><p>MSS rectal cancer appears to represent a heterogeneous group of tumors that may be categorized both on the basis of CIN status and telomere maintenance mechanism. MSS CIN- rectal cancers appear to have longer telomeres than those of MSS CIN+ rectal cancers and to utilize ALT rather than activation of telomerase. </p> </div

Telomere length in tumor DNA based on CIN status and telomerase activation.

<div><p>Panel A: The telomere length of tumor DNA in chromosomally stable (CIN-) rectal cancer is significantly longer than that of chromosomally unstable (CIN+) rectal cancer (p=0.0066). CIN status is determined as CIN- if from <20% of clones show DNA copy number gains or losses. Rectal cancer is CIN+ if more than 20% of clones have gains or losses. </p> <p>Panel B: Activation of telomerase (Telomerase +) in rectal cancer correlates with shorter tumor telomere length than in tumors that do not utilize telomerase (Telomerase -) as a telomere maintenance mechanism (p=0.0040).</p></div