Functional telomeres are required for the replicability of cancer cells. The G-rich strand of telomeric DNA can fold into a 4-stranded structure known as the G-quadruplex (G4), whose stabilization alters telomere function limiting cancer cell growth. Therefore, the G4 ligand RHPS4 may possess antitumor activity. Here, we show that RHPS4 triggers a rapid and potent DNA damage response at telomeres in human transformed fibroblasts and melanoma cells, characterized by the formation of several telomeric foci containing phosphorylated DNA damage response factors γ-H2AX, RAD17, and 53BP1. This was dependent on DNA repair enzyme ATR, correlated with delocalization of the protective telomeric DNA-binding protein POT1, and was antagonized by overexpression of POT1 or TRF2. In mice, RHPS4 exerted its antitumor effect on xenografts of human tumor cells of different histotype by telomere injury and tumor cell apoptosis. Tumor inhibition was accompanied by a strong DNA damage response, and tumors overexpressing POT1 or TRF2 were resistant to RHPS4 treatment. These data provide evidence that RHPS4 is a telomere damage inducer and that telomere disruption selectively triggered in malignant cells results in a high therapeutic index in mice. They also define a functional link between telomere damage and antitumor activity and reveal the key role of telomere-protective factors TRF2 and POT1 in response to this anti-telomere strategy.
Supplementation of patients receiving cisplatin chemotherapy with vitamin E decreases the incidence and severity of peripheral neurotoxicity.
Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce a specific recruitment of Che-1 on the TP53 and p21 promoters. Interestingly, it has a profound effect on the basal expression of p53, which is preserved following DNA damage. Notably, Che-1 contributes to the maintenance of the G2/M checkpoint induced by DNA damage. These findings identify a mechanism by which checkpoint kinases regulate responses to DNA damage.
Dysfunctional telomeres suppress tumour progression by activating cell-intrinsic programs that lead to growth arrest. Increased levels of TRF2, a key factor in telomere protection, are observed in various human malignancies and contribute to oncogenesis. We demonstrate here that a high level of TRF2 in tumour cells decreased their ability to recruit and activate natural killer (NK) cells. Conversely, a reduced dose of TRF2 enabled tumour cells to be more easily eliminated by NK cells. Consistent with these results, a progressive upregulation of TRF2 correlated with decreased NK cell density during the early development of human colon cancer. By screening for TRF2-bound genes, we found that HS3ST4--a gene encoding for the heparan sulphate (glucosamine) 3-O-sulphotransferase 4--was regulated by TRF2 and inhibited the recruitment of NK cells in an epistatic relationship with TRF2. Overall, these results reveal a TRF2-dependent pathway that is tumour-cell extrinsic and regulates NK cell immunity.
About half of cancers sustain mutations in the TP53 gene, whereas the other half maintain a wild-type p53 (wtp53) but may compromise the p53 response because of other alterations. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of p53 oncosuppressor function. Here, we show, by microarray analysis, that wtp53 lost the target gene activation following stable knockdown of HIPK2 (HIPK2i) in colon cancer cell line. Our data show that the stable knockdown of HIPK2 led to wtp53 misfolding, as detected by p53 immunoprecipitation with conformationspecific antibodies, and that p53 protein misfolding impaired p53 DNA binding and transcription of target genes. We present evidence that zinc supplementation to HIPK2i cells increased p53 reactivity to conformation-sensitive PAb1620 (wild-type conformation) antibody and restored p53 sequence-specific DNA binding in vivo and transcription of target genes in response to Adriamycin treatment. Finally, combination of zinc and Adriamycin suppressed tumor growth in vivo and activated misfolded p53 that induced its target genes in nude mice tumor xenografts derived from HIPK2i cells. Bioinformatics analysis of microarray data from colon cancer patients showed significant association of poor survival with low HIPK2 expression only in tumors expressing wtp53. These results show a critical role of HIPK2 in maintaining the transactivation activity of wtp53 and further suggest that low expression of HIPK2 may impair the p53 function in tumors harboring wtp53.
Bcl‐2 protein has been shown to contribute to oncogenesis because it can transform and immortalize cells in cooperation with c‐myc, ras, or viral genes. However, in vivo studies have not yet established whether bcl‐2 can play a role in metastasis. Here we investigate the potential metastatic role of bcl‐2. We introduced the human bcl‐2 gene into a low bcl‐2 expressing human breast cancer cell line MCF7 ADR. We demonstrate that two bcl‐2 overexpressing clones injected intravenously or intramuscularly into nude mice induce a significantly higher number of experimental and spontaneous lung metastases compared to the control transfectant clone. We demonstrate that bcl‐2 overexpressing clones are more invasive and migratory in response to chemotactic stimuli than the control transfectant clone. Furthermore, zymographic analysis shows that secretion of 72 and 92 kDa gelatinases increases in the two bcl‐2 overexpressing transfectants. Tumors originating from bcl‐2 overexpressing clones also show a decrease in the latency period of tumor appearance. In conclusion, our data show that bcl‐2 overexpression enhances both tumorigenicity and metastatic potential of MCF7 ADR cells by inducing metastasis‐associated properties.—Del Bufalo, D., Biroccio, A., Leonetti, C., Zupi, G. Bcl‐2 overexpression enhances the metastatic potential of a human breast cancer line. FASEB J. 11, 947–953 (1997)
This study had two goals: 1) to evaluate the biological effect of the novel pentacyclic acridine 3, 11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) on human melanoma lines possessing long telomeres, and 2) to elucidate the relationship between G-quadruplex-based telomerase inhibitor-induced cellular effects and telomere length/dysfunction. The cellular pharmacological effects of RHPS4 have been evaluated by treating melanoma lines with increasing concentrations of RHPS4. A dose-dependent inhibition of cell proliferation was observed in all the lines during short-term treatment. Flow cytometric analysis demonstrated that RHPS4 induced a dose-dependent accumulation of cells in the S-G 2 /M phase of cell cycle. The RHPS4-induced cell cycle alteration was irreversible even at low doses, and the cells died from apoptosis. At high RHPS4 concentration, apoptosis was accompanied by the induction of a senescence phenotype: large cell size, vacuolated cytoplasm, and -galactosidase activity. The shortterm biological activity of RHPS4 was not caused by telomere shortening, but it was associated with telomere dysfunction, in terms of presence of telomeric fusions, polynucleated cells, and typical images of telophase bridge. In conclusion, our results demonstrate that the G-quadruplex ligand RHPS4 can function in a telomere length-independent manner through its ability to cause telomere-capping alteration.
A novel antibody-drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt-Pt-Ep1). The HFt-Pt-Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4(+) melanoma cell line, but not to a CSPG4(-) breast carcinoma cell line. As compared to the cisplatin-containing ferritin nanoparticle alone (HFt-Pt), which inhibited thymidine incorporation more efficiently in breast carcinoma than melanoma cells, the mAb-derivatized HFt-Pt-Ep1 nanoparticle had a 25-fold preference for the latter. A similar preference for melanoma was observed upon systemic intravenous administration of HFt-Pt-Ep1 to nude mice xenotransplanted with pre-established, palpable melanoma and breast carcinoma tumors. Thus, we have been able to determine precise combinations and stoichiometric relationships between mAbs and nanoparticle protein cages, whereby the latter lose their tropism for ubiquitously distributed cellular receptors, and acquire instead remarkably lineage-selective binding. HFt-Pt-Ep1 is therefore an interesting model to improve the therapeutic index of antiblastic therapy in a tumor such as melanoma, which at its advanced stages is totally refractory to mono- and combination-chemotherapy.
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