A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes

BACKGROUND: The antihypertensive compound hydralazine is a known demethylating agent. This phase I study evaluated the tolerability and its effects upon DNA methylation and gene reactivation in patients with untreated cervical cancer. METHODS: Hydralazine was administered to cohorts of 4 patients at the following dose levels: I) 50 mg/day, II) 75 mg/day, III) 100 mg/day and IV) 150 mg/day. Tumor biopsies and peripheral blood samples were taken the day before and after treatment. The genes APC, MGMT; ER, GSTP1, DAPK, RARβ, FHIT and p16 were evaluated pre and post-treatment for DNA promoter methylation and gene expression by MSP (Methylation-Specific PCR) and RT-PCR respectively in each of the tumor samples. Methylation of the imprinted H19 gene and the "normally methylated" sequence clone 1.2 was also analyzed. Global DNA methylation was analyzed by capillary electrophoresis and cytosine extension assay. Toxicity was evaluated using the NCI Common Toxicity Criteria. RESULTS: Hydralazine was well tolerated. Toxicities were mild being the most common nausea, dizziness, fatigue, headache and palpitations. Overall, 70% of the pretreatment samples and all the patients had at least one methylated gene. Rates of demethylation at the different dose levels were as follows: 50 mg/day, 40%; 75 mg/day, 52%, 100 mg/day, 43%, and 150 mg/day, 32%. Gene expression analysis showed only 12 informative cases, of these 9 (75%) re-expressed the gene. There was neither change in the methylation status of H19 and clone 1.2 nor changes in global DNA methylation. CONCLUSION: Hydralazine at doses between 50 and 150 mg/day is well tolerated and effective to demethylate and reactivate the expression of tumor suppressor genes without affecting global DNA methylatio

Serum nucleosomes during neoadjuvant chemotherapy in patients with cervical cancer. Predictive and prognostic significance

BACKGROUND: It has been shown that free DNA circulates in serum plasma of patients with cancer and that at least part is present in the form of oligo- and monucleosomes, a marker of cell death. Preliminary data has shown a good correlation between decrease of nucleosomes with response and prognosis. Here, we performed pre- and post-chemotherapy determinations of serum nucleosomes with an enzyme-linked immunosorbent assay (ELISA) method in a group of patients with cervical cancer receiving neoadjuvant chemotherapy. METHODS: From December 2000 to June 2001, 41 patients with cervical cancer staged as FIGO stages IB2-IIIB received three 21-day courses of carboplatin and paclitaxel, both administered at day 1; then, patients underwent radical hysterectomy. Nucleosomes were measured the day before (baseline), at day seven of the first course and day seven of the third course of chemotherapy. Values of nucleosomes were analyzed with regard to pathologic response and to time to progression-free and overall survival. RESULTS: All patients completed chemotherapy, were evaluable for pathologic response, and had nucleosome levels determined. At a mean follow-up of 23 months (range, 7–26 months), projected progression time and overall survival were 80.3 and 80.4%, respectively. Mean differential values of nucleosomes were lower in the third course as compared with the first course (p >0.001). The decrease in the third course correlated with pathologic response (p = 0.041). Survival analysis showed a statistically significant, better progression-free and survival time in patients who showed lower levels at the third course (p = 0.0243 and p = 0.0260, respectively). Cox regression analysis demonstrated that nucleosome increase in the third course increased risk of death to 6.86 (95% confidence interval [CI 95%], 0.84–56.0). CONCLUSION: Serum nucleosomes may have a predictive role for response and prognostic significance in patients with cervical cancer patients treated with neoadjuvant chemotherapy

Cancer progression mediated by horizontal gene transfer in an in vivo model.

It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy

Tumorigenesis and DNA transfer in recipient murine NIH3T3 cells after passive transfection. A.

<p>Tumor growth in nude mice from “passively” transformed cells. Faster and higher tumor growth was observed in SB1 pool and CCPS pool (NIH3T3 exposed to supernatant of SW480 cells and to the serum of a patient with colon cancer, respectively). SW480 cells were used as positive control, whereas NIH3T3 and NIH3T3 exposed to normal serum showed essentially no growth<b>. B.</b> Representative pictures of tumors in mice from each group. <b>C.</b> Southern blot hybridization of SB1 and CCPS pools of cells against genomic DNA of SW480 cells. Lane SW480 cells are the positive control and NIH3T3, the negative one. A clear hybridization signal is only observed in SB1 and CCPS lanes. <b>D.</b> FISH analysis of repetitive human sequences. Positive control is human lymphocytes and murine cells negative control. SB1 cells shows strong signal. <b>E.</b> Tumor growth is similar in NIH3T3 actively transfected with genomic DNA from SW480 cells (Neo-Geno) and actively transfected DNA extracted from supernatant of SW480 cells as compared with no growth in NIH3T3 (-Crt) and transfected with the empty-vector only. Positive control, SW480 cells.</p

Human DNA transfer in rat colon tumors by PCR-sequencing.

<p>Representative pictures of PCR detection of a repetitive sequence of rat (<i>LINE 1</i>) in a rat tumor (DMH and DMH+SW480). (<b>A</b>) <i>Alu Yd6</i> human sequences were only amplified from the colon tumors of DMH+SW480-treated rats. Rat tail and human cells were used as positive and negative controls. Human <i>K-ras</i> and <i>RAB30</i> genes were only detected in the tumors of rats receiving DMH and SW480 cells. (<b>B</b>) Sequence analysis of the PCR product of <i>RAB30</i> in a colon tumor treated with DMH+SW480 cells. Arrows indicate the position where the nucleotide sequence is different between species and clearly shows the existence of both sequences. Human SW480 cells (control).</p

DNA copy number analysis of extracellular and intracellular DNA from SW480 cells and gene transfer to murine NIH3T3 cells. A.

<p>Heat map representing the DNA copy number along chromosome 8. Blue represents regions with deletions and red regions with amplifications. A nearly identical pattern of DNA copy number changes between extracellular (SpDNA SW480) and intracellular (DNA SW480) DNA, compared to a common normal reference can be observed. RT-PCR, PCR and sequencing of Human <i>K-ras</i> (<b>B</b>) and <i>RAB30</i> (<b>C</b>)<b>.</b> Negative control was NIH3T3 cells.</p

Tumorigenesis after “active” transfection with DNA supernatant of SW480 cells and passive transfection using DNA-depleted supernatant. A.

<p>Agarose gel electrophoresis of DNA extracted from supernatant (Sp), DNA from supernatant treated with DNAse I (Sp+D), protease only (Sp+P), and both (Sp+D+P). DNA is partially degraded by DNAse I and protease, but fully degraded when exposed to both treatments. <b>B.</b> No tumor growth was observed in NIH3T3 exposed to DNA-depleted (DNAse I/Prot) supernatant of SW480 cells, while passively transfected NIH3T3 with untreated supernatant are tumorigenic. +Ctr are SW480 cells and −Ctr are NIH3T3 cells.</p