Completion of excision repair patches in human cell preparations: identification of a probable mode of excision and resynthesis

Excision repair of u.v. damage in human fibroblasts is moresensitive to inhibitors of DNA polymerase (cytosine arabinoside,aphidicolin) than to an inhibitor of polymerase ß(dideoxythymidine), which indicates a greater role in repairfor polymerase than for polymerase ß. These inhibitorsall generate shortened patches with free 3' termini; the detailedstructure of these patches was investigated in permeable cellsor isolated nuclei by degradation of DNA with exonuclease IIIand by resynthesis with DNA polymerase I (Klenow fragment) andT4 DNA ligase. The structure of the shortened patches appearsto be a short stretch of DNA synthesized in the 5'3' directionwithin a longer single-strand gap. The single-strand gap aheadof the 3' terminus can be bridged only by the combined actionof polymerase and ligase. This structure implies that excisionmust involve removal of an oligonucleotide or widening of agap by 5'3' exonuclease action to produce a single-strand regionwide enough to be a substrate for polymerase . There is no evidencefor structures generated by nick translation or strand displacement.     

Inhibition of malignant transformation in vitro by inhibitors of poly(ADP-ribose) synthesis.

Malignant transformation in vitro of hamster embryo cells and mouse C3H 10T 1/2 cells by x-rays, ultraviolet light, and chemical carcinogens was inhibited by benzamide and by 3-aminobenzamide at concentrations that are specific for inhibition of poly(ADP-ribose) formation. These compounds slow the ligation stage of repair of x-ray and alkylation damage but not of ultraviolet light damage. At high concentrations they also inhibited de novo synthesis of DNA purines and DNA methylation by S-adenosylmethionine. The suppression of transformation by the benzamides is in striking contrast to their reported effectiveness in enhancing sister chromatid exchange, mutagenesis, and killing in cells exposed to alkylating agents. Our results suggest that mechanisms regulating malignant transformation are different from those regulating DNA repair, sister chromatid exchange, and mutagenesis and may be associated with changes in gene regulation and expression caused by alterations in poly(ADP-ribosyl)ation.     

Integration of repulsive guidance cues generates avascular zones that shape mammalian blood vessels

RATIONALE: Positive signals, such as vascular endothelial growth factor, direct endothelial cells (ECs) to specific locations during blood vessel formation. Less is known about repulsive signal contribution to shaping vessels. Recently, "neuronal guidance cues" have been shown to influence EC behavior, particularly in directing sprouting angiogenesis by repelling ECs. However, their role during de novo blood vessel formation remains unexplored. Objective: To identify signals that guide and pattern the first mammalian blood vessels. MethODS AND RESULTS: Using genetic mouse models, we show that blood vessels are sculpted through the generation of stereotyped avascular zones by EC-repulsive cues. We demonstrate that Semaphorin3E (Sema3E) is a key factor that shapes the paired dorsal aortae in mouse, as sema3E(-/-) embryos develop an abnormally branched aortic plexus with a markedly narrowed avascular midline. In vitro cultures and avian grafting experiments show strong repulsion of ECs by Sema3E-expressing cells. We further identify the mouse notochord as a rich source of multiple redundant neuronal guidance cues. Mouse embryos that lack notochords fail to form cohesive aortic vessels because of loss of the avascular midline, yet maintain lateral avascular zones. We demonstrate that lateral avascular zones are directly generated by the lateral plate mesoderm, a critical source of Sema3E. CONCLUSIONS: These findings demonstrate that Sema3E-generated avascular zones are critical regulators of mammalian cardiovascular patterning and are the first to identify a repulsive role for the lateral plate mesoderm. Integration of multiple, and in some cases redundant, repulsive cues from various tissues is critical to patterning the first embryonic blood vessels.     

Targeting protein-trafficking pathways alters melanoma treatment sensitivity

Protein-trafficking pathways are targeted here in human melanoma cells using methods independent of oncogene mutational status, and the ability to up-regulate and down-regulate tumor treatment sensitivity is demonstrated. Sensitivity of melanoma cells to cis-diaminedichloroplatinum II (cDDP, cis-platin), carboplatin, dacarbazine, or temozolomide together with velaparib, an inhibitor of poly (ADP ribose) polymerase 1, is increased by up to 10-fold by targeting genes that regulate both protein trafficking and the formation of melanosomes, intracellular organelles unique to melanocytes and melanoma cells. Melanoma cells depleted of either of the protein-trafficking regulators vacuolar protein sorting 33A protein (VPS33A) or cappuccino protein (CNO) have increased nuclear localization of cDDP, increased nuclear DNA damage by platination, and increased apoptosis, resulting in increased treatment sensitivity. Depleted cells also exhibit a decreased proportion of intracellular, mature melanosomes compared with undepleted cells. Modulation of protein trafficking via cell-surface signaling by binding the melanocortin 1 receptor with the antagonist agouti-signaling protein decreased the proportion of mature melanosomes formed and increased cDDP sensitivity, whereas receptor binding with the agonist melanocyte-stimulating hormone resulted in an increased proportion of mature melanosomes formed and in decreased sensitivity (i.e., increased resistance) to cDDP. Mutation of the protein-trafficking gene Hps6, known to impair the formation of mature melanosomes, also increased cDDP sensitivity. Together, these results indicate that targeting protein-trafficking molecules markedly increases melanoma treatment sensitivity and influences the degree of melanosomes available for sequestration of therapeutic agents.     

Recapitulation of the cellular xeroderma pigmentosum-variant phenotypes using short interfering RNA for DNA polymerase H

The lesion-specific DNA polymerase POLH gene is mutated in xeroderma pigmentosum variant (XP-V) patients who exhibit an increased skin cancer incidence from UV exposure. Normal cells in which POLH expression was reduced using short interfering RNAs (siRNAs) were compared with the XP-V cellular phenotype that results from naturally occurring inactivating mutations. Stable clones expressing siRNA had partially reduced POLH protein levels, and intermediate levels of UV sensitivity and S phase checkpoint activation, but similar levels of Mre11 foci as in XP-V cells. Therefore, suppression of POLH expression levels by siRNA recapitulates most of the phenotypes seen in cells from XP-V patients with inactivating mutations in POLH.     

Quality assurance and iron deficiency in Egypt.

OBJECTIVE: To develop an intervention in rural Egypt to address the problem of iron deficiency anemia and to demonstrate the effectiveness of applying quality assurance (QA) methods in combating this. DESIGN: Assessment of an intervention study utilizing QA methods. SETTING: Rural primary care clinics in Egypt. STUDY PARTICIPANTS: One hundred and eighty pregnant mothers (and their 180 children) were randomly selected at two clinic sites from all those who were diagnosed as having an iron deficiency disorder. INTERVENTION: Multi-disciplinary teams were formed to develop and deliver health promotional approaches related to iron deficiency to the study participants. By using QA techniques the teams were able to strengthen local capacity and participant compliance to the educational messages. MAIN OUTCOME MEASURES: Pre- and post-measurements of client satisfaction, results of hemoglobin lab tests, and the extent of retention of nutritional messages by the participants. RESULTS: Eighty percent of the study population demonstrated satisfactory knowledge of the nutritional messages. There was a 75% improvement of client satisfaction with the clinic and an effective follow-up system of care was designed and implemented successfully for each clinic. On average, the number of children aged less than 5 years diagnosed with an iron deficiency disorder decreased from 37% to 5%. Similar success was achieved with the pregnant mothers: the prevalence of iron deficiency anemia was reduced from 100% to only 14%. CONCLUSIONS: The use of QA process improvement techniques was extremely effective in reducing iron deficiency anemia among the target population. There is an increasing need to include quality methods in micronutrient intervention techniques.