Control of DNA structure and function by phytochemicals/DNA interaction: Resveratrol/piceatannol induces Cu2+-independent, cleavage of supercoiled plasmid DNA.

Topoisomerases are enzymes that catalyze DNA unwinding and scissions to resolve topological entanglements possibly arising during DNA replication/transcription. Chemicals which disrupt or inhibit topoisomerase-mediated DNA unwinding can induce breaks that subsequently lead to programmed cell death. Herein we perform experiments guided by the following considerations. First, topoisomerase 1 initiates DNA cleavage utilizing the hydroxyl group of tyrosine 723 on its catalytic site as a nucleophile to attack the electrophilic phosphate on the DNA sugar-phosphate backbone. Secondly, the grape polyphenol resveratrol displays both topoisomerase inhibitory and Cu2+-dependent DNA-cutting activities, which contribute to its DNA replication/transcription inhibitory/anti-tumorigenic effects. Lastly, resveratrol contains a tyrosine-like phenolic ring; thus, upon binding to DNA whether resveratrol could act as a tyrosine mimetic to unwind and cut DNA via its hydroxyl groups warrants investigation. Polyphenol-DNA interactions (PDIs) were investigated using UV-visible spectral analysis; additionally, PDI mediated DNA changes were further analyzed by agarose gel electrophoresis using 3 supercoiled plasmid DNAs (pBR322, pSJ3, pHOT-1) as substrates. Resveratrol mediates time- and temperature-dependent, Cu2+-independent, non-enzymatic cleavage of supercoiled plasmid DNA into open, circular DNA products. Varying degree of unwinding of supercoiled DNA nucleolytic activity was also observed with other polyphenols including, piceatannol, quercetin, myricetin and EGCG. Interestingly, we found that piceatannol mediated Cu2+-independent DNA-cleavage activity was abolished by EDTA. The PDI-mediated nucleolytic cleavage of supercoiled DNA reported herein shows that polyphenolic phytochemicals display genome-active, nuclear effects by directly targeting the DNA topology which in turn could impact macromolecular processes associated with faithful replication and transmission of genetic information.

Loss of the p12 subunit of DNA polymerase delta leads to a defect in HR and sensitization to PARP inhibitors.

Human DNA polymerase δ is normally present in unstressed, non-dividing cells as a heterotetramer (Pol δ4). Its smallest subunit, p12, is transiently degraded in response to UV damage, as well as during the entry into S-phase, resulting in the conversion of Pol δ4 to a trimer (Pol δ3). In order to further understand the specific cellular roles of these two forms of Pol δ, the gene (POLD4) encoding p12 was disrupted by CRISPR/Cas9 to produce p12 knockout (p12KO) cells. Thus, Pol δ4 is absent in p12KO cells, leaving Pol δ3 as the sole source of Pol δ activity. GFP reporter assays revealed that the p12KO cells exhibited a defect in homologous recombination (HR) repair, indicating that Pol δ4, but not Pol δ3, is required for HR. Expression of Flag-tagged p12 in p12KO cells to restore Pol δ4 alleviated the HR defect. These results establish a specific requirement for Pol δ4 in HR repair. This leads to the prediction that p12KO cells should be more sensitive to chemotherapeutic agents, and should exhibit synthetic lethal killing by PARP inhibitors. These predictions were confirmed by clonogenic cell survival assays of p12KO cells treated with cisplatin and mitomycin C, and with the PARP inhibitors Olaparib, Talazoparib, Rucaparib, and Niraparib. The sensitivity to PARP inhibitors in H1299-p12KO cells was alleviated by expression of Flag-p12. These findings have clinical significance, as the expression levels of p12 could be a predictive biomarker of tumor response to PARP inhibitors. In addition, small cell lung cancers (SCLC) are known to exhibit a defect in p12 expression. Analysis of several SCLC cell lines showed that they exhibit hypersensitivity to PARP inhibitors, providing evidence that loss of p12 expression could represent a novel molecular basis for HR deficiency.

Two forms of human DNA polymerase δ: Who does what and why?

DNA polymerase δ (Pol δ) plays a central role in lagging strand DNA synthesis in eukaryotic cells, as well as an important role in DNA repair processes. Human Pol δ4 is a heterotetramer of four subunits, the smallest of which is p12. Pol δ3 is a trimeric form that is generated in vivo by the degradation of the p12 subunit in response to DNA damage, and during entry into S-phase. The biochemical properties of the two forms of Pol δ, as well as the changes in their distribution during the cell cycle, are reviewed from the perspective of understanding their respective cellular functions. Biochemical and cellular studies support a role for Pol δ3 in gap filling during DNA repair, and in Okazaki fragment synthesis during DNA replication. Recent studies of cells in which p12 expression is ablated, and are therefore null for Pol δ4, show that Pol δ4 is not required for cell viability. These cells have a defect in homologous recombination, revealing a specific role for Pol δ4 that cannot be performed by Pol δ3. Pol δ4 activity is required for D-loop displacement synthesis in HR. The reasons why Pol δ4 but not Pol δ3 can perform this function are discussed, as well as the question of whether helicase action is needed for efficient D-loop displacement synthesis. Pol δ4 is largely present in the G1 and G2/M phases of the cell cycle and is low in S phase. This is discussed in relation to the availability of Pol δ4 as an additional layer of regulation for HR activity during cell cycle progression.

Perforation of the duodenum by an ingested toothbrush.

We report a rare case of duodenal perforation caused by an ingested 12-cm long toothbrush handle. A 22-year-old female presented with intermittent epigastric pain for 6 d after swallowing a broken toothbrush. The swallowed toothbrush could not be removed from the second portion of the duodenum by endoscopy. Laparotomy revealed a perforation in the anterior wall of the duodenal bulb. The toothbrush was removed via the perforation which was debrided and closed. There were no postoperative complications.

Perforation through small bowel malignant tumors.

Data on 19 patients (6 women and 13 men) with malignancy perforation through small bowel tissue were retrospectively reviewed. The median patient age was 57 years (range, 41-81 years). The histopathology included lymphoma (seven patients), leiomyosarcoma (two patients), gastrointestinal stromal tumor (one patient), adenocarcinoma (one patient), metastatic carcinomas with unknown primary tumor (four patients), metastatic adenocarcinoma from the lung (one patient), and metastatic carcinomas from the hypopharynx (one patient), cervix (one patient), and lung (one patient). Resection of a segment of perforated bowel with primary anastomosis was performed in 16 patients, wedge resection of perforated lesion with plication in two patients, and loop ileostomy in one patient. Postoperative deaths occurred in 10 (52.6%) patients, owing to sepsis and organ functional failure. Seven patients died from the primary malignancy at a median follow-up of 6.5 months (range, 5 months to 1 year 9 months) after surgery. Moreover, two patients with small bowel lymphoma were alive with disease at 4 years 8 months and 7 years 1 month after surgery. In conclusion, perforation through small bowel malignant tumors had a high postoperative mortality rate. High index of suspicion of the disease with early surgical treatment may improve treatment outcomes.

Sonographic features of breast hamartomas.

OBJECTIVE: The purpose of this study was to elucidate the sonographic characteristics of breast hamartomas. METHODS: Data and sonographic images of 14 breast hamartomas were retrospectively reviewed. RESULTS: All patients had clinically palpable lumps. The median patient age was 39.5 years (range, 24-60 years). Eleven (78.6%) tumors occurred in the right breast, and 3 (21.4%) were in the left. The median tumor size measured by sonography was 2.8 cm (range, 1.2-4.9 cm). The median longest transverse dimension-anteroposterior diameter ratio of the tumors was 2.44 (range, 1.52-3.73). All tumors were oval and compressible with transducer pressure. Thirteen (92.9%) tumors were well circumscribed with smooth tumor margins, and 1 (7.1%) had indistinct margins. The internal echo texture was hyperechoic in 6 (42.9%), mixed (heterogeneous) echogenicity in 5 (35.7%), and isoechoic in 3 (21.4%). Four (28.6%) tumors had echogenic halos, and 2 (14.3%) had anechoic halos. Ten (71.4%) tumors had no retrotumor acoustic phenomena. Two (14.3%) had bilateral edge shadowing; 1 (7.1%) had posterior enhancement; and 1 (7.1%) had a mixture of enhancement and shadowing. CONCLUSIONS: Breast hamartomas were well-circumscribed, solid, oval tumors without intratumor microcalcification. The internal echo texture of most hamartomas is either hyperechoic or composed of mixed echogenicity. Retrotumor acoustic phenomena were absent in most hamartomas.

Surgical treatment of solitary thyroid nodules via fine-needle aspiration biopsy and frozen-section analysis.

BACKGROUND: Fine-needle aspiration biopsy (FNAB) and frozen-section analysis of managing solitary thyroid nodules continue to generate considerable controversy. METHODS: This study was a retrospective review of 619 patients with solitary thyroid nodules who underwent thyroidectomy. RESULTS: Of 540 FNABs, 35 (6.5%) were positive for malignancy, 276 (51.1%) were benign, and 229 (42.4%) were suspicious. Only 5.1% were false negative, and 11.4% were false positive. Diagnostic FNAB sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for malignancy were 86.1%, 59.7%, 33.0%, 94.9%, and 64.6%, respectively. Of 569 patients analyzed by frozen section, diagnosis was deferred in 86 (15.1%) patients, and results were positive for malignancy in 92 (16.2%) and benign in 391 (68.7%). No false-positive results were noted, but 2.3% (391) were false negative. Of 86 deferred frozen sections, 11 (12.8%) patients had malignant tumors confirmed by permanent section. Diagnostic frozen-section sensitivity, specificity, PPV, NPV, and accuracy for carcinoma were 82.1%, 100%, 100%, 95.8%, and 96.5%, respectively. Sensitivity, specificity, PPV, NPV, and accuracy for frozen-section analysis for diagnosis of carcinoma in patients with suspicious FNAB were 83.9%, 100%, 100%, 94.9%, and 96.0%, respectively. CONCLUSIONS: FNAB is a sensitive diagnostic modality in selecting patients who require surgery. Routine use of frozen-section analysis is unwarranted for benign FNAB results. Frozen section is specific and cost-effective in determining the extent of surgery in patients with suspicious or malignant FNABs.