Analytical Cellular Pathology - Volume 30, issue 5

Authors: Thangasamy, Thilakavathy | Sittadjody, Sivanandane | H. Limesand, Kirsten | Burd, Randy

Article Type: Research Article

Abstract: Dacarbazine (DTIC) has been used for the treatment of melanoma for decades. However, monotherapy with this chemotherapeutic agent results only in moderate response rates. To improve tumor response to DTIC current clinical trials in melanoma focus on combining a novel targeted agent with chemotherapy. Here, we demonstrate that tyrosinase which is commonly overexpressed in melanoma activates the bioflavonoid quercetin (Qct) and promotes an ataxia telangiectasia mutated (ATM)-dependent DNA damage response. This response sensitizes melanoma cells that overexpress tyrosinase to DTIC. In DB-1 melanoma cells that overexpress tyrosinase (Tyr+ cells), the threshold for phosphorylation of ATM and p53 at serine …15 was observed at a low dose of Qct (25 μM) when compared to the mock transfected pcDNA3 cells, which required a higher dose (75 μM). Both pcDNA3 and Tyr+ DB-1 cells demonstrated similar increases in phosphorylation of p53 at other serine sites, but in the Tyr+ cells, DNApk expression was found to be reduced compared to control cells, indicating a shift towards an ATM-mediated response. The DB-1 control cells were resistant to DTIC, but were sensitized to apoptosis with high dose Qct, while Tyr+ cells were sensitized to DTIC with low or high dose Qct. Qct also sensitized SK Mel 5 (p53 wildtype) and 28 (p53 mutant) cells to DTIC. However, when SK Mel 5 cells were transiently transfected with tyrosinase and treated with Qct plus DTIC, SK Mel 5 cells demonstrated a more than additive induction of apoptosis. Therefore, this study demonstrates that tyrosinase overexpression promotes an ATM-dependent p53 phosphorylation by Qct treatment and sensitizes melanoma cells to dacarbazine. In conclusion, these results suggest that Qct or Qct analogues may significantly improve DTIC response rates in tumors that express tyrosinase. Show more

Keywords: Tyrosinase, melanoma, p53, quercetin, ATM, DTIC

DOI: 10.3233/CLO-2008-0441

Citation: Analytical Cellular Pathology, vol. 30, no. 5, pp. 371-387, 2008

Authors: Pinto, Mafalda | Vieira, Joana | Ribeiro, Franclim R. | Soares, Maria J. | Henrique, Rui | Oliveira, Jorge | Jerónimo, Carmen | Teixeira, Manuel R.

Article Type: Research Article

Abstract: Background: A defective mitotic checkpoint has been proposed to contribute to chromosomal instability (CIN). We have previously shown that expression changes of the mitotic arrest deficiency (MAD) gene family plays a role in renal cell cancer (RCC) characterized by numerical chromosomal changes, namely papillary and chromophobe carcinomas, but nothing is known about the expression of mitotic checkpoint genes in the clear cell histotype (ccRCC). Methods: We analyzed the mRNA expression levels of the major mitotic checkpoint genes of the budding uninhibited by benzimidazole family (BUB1, BUBR1, BUB3) and of the MAD gene family (MAD1, MAD2L1, MAD2L2) by real-time quantitative …PCR in 39 ccRCC and in 36 normal kidney tissue samples. We have additionally analyzed these tumors by comparative genomic hybridization (CGH) in order to evaluate the relationship between mitotic checkpoint defects and the pattern of chromosome changes in this subset of RCC. Results: BUB1, BUBR1, MAD1 and MAD2L1 showed significant expression differences in tumor tissue compared to controls (BUB1, BUBR1 and MAD2L1 were overexpressed, whereas MAD1 was underexpressed). Overexpression of BUB1 and BUBR1 was significantly correlated with the number of genomic copy number changes (p<0.001 for both genes) and with Furhman grade of the tumors (p=0.006 and p=0.005, respectively). Conclusions: We conclude that BUB1 and BUBR1 overexpression plays a role in cytogenetic and morphologic progression of ccRCC. Show more

Keywords: Clear cell renal cell carcinoma (ccRCC), mitotic checkpoint, gene expression, BUB, MAD

DOI: 10.3233/CLO-2008-0439

Citation: Analytical Cellular Pathology, vol. 30, no. 5, pp. 389-395, 2008

Authors: Groot, Arjan J. | Gort, Eelke H. | van der Wall, Elsken | van Diest, Paul J. | Vooijs, Marc

Article Type: Research Article

Abstract: Hypoxia is a hallmark of solid cancers and triggers the transcription of genes responsible for cell survival. The transcription factor Hypoxia-Inducible Factor 1 (HIF-1) is a key regulator in this response and frequently activated in human cancer. HIF-1 activation is associated with tumor aggressiveness and poor clinical outcome and, therefore, may provide an attractive therapeutic target. Here we provide a novel approach for HIF-1 targeted therapy using single-domain llama antibodies directed against the HIF-1α oxygen dependent degradation domain which encompass the N-terminal transactivation domain. Conditional expression of HIF intrabodies in mammalian cells interfered with binding to pVHL and inhibited hypoxia …induced activation of endogenous target genes. Inducible intrabody targeting is a highly specific strategy for temporal protein inactivation and may have applications for disease treatment. Show more

Keywords: Cancer, HIF, hypoxia, intrabody, single-chain antibody fragment, VHH

DOI: 10.3233/CLO-2008-0442

Citation: Analytical Cellular Pathology, vol. 30, no. 5, pp. 397-409, 2008

Authors: Zache, Nicole | Lambert, Jeremy M.R. | Wiman, Klas G. | Bykov, Vladimir J.N.

Article Type: Research Article

Abstract: Reactivation of the tumor suppressor activity to mutant p53 should trigger massive apoptosis and eliminate tumors. The low molecular weight compounds PRIMA-1 and the structural analog PRIMA-1MET reactivate human mutant p53 in vitro and suppress growth of human tumor xenografts in SCID mice. However, little is known about their effect on mouse mutant p53 in mouse tumor cells. We have examined the effect of PRIMA-1MET on mouse sarcomas, mammary carcinomas and chemically induced fibrosarcomas. PRIMA-1MET showed potent growth suppression in mutant p53-carrying mouse tumors in vitro and a significant anti-tumor effect in syngeneic mice in vivo. These …results demonstrate that PRIMA-1MET targets mouse tumors carrying mutant p53 and provide strong support for the anti-tumor efficiency of PRIMA-1MET in vivo. Show more

Keywords: Mutant p53, PRIMA-1^MET, mouse tumors, syngeneic mouse model, cancer therapy

DOI: 10.3233/CLO-2008-0440

Citation: Analytical Cellular Pathology, vol. 30, no. 5, pp. 411-418, 2008

Authors: Heikaus, Sebastian | van den Berg, Linda | Kempf, Tobias | Mahotka, Csaba | Gabbert, Helmut Erich | Ramp, Uwe

Article Type: Research Article

Abstract: Renal cell carcinomas (RCCs) exhibit a marked resistance towards apoptosis. Although most apoptotic stimuli converge at the level of the mitochondria, little is known about the mitochondrial apoptosis pathway in renal cell carcinomas. The aim of the present study, therefore, was to investigate the functionality of the mitochondrial apoptosis pathway in renal cell carcinoma cell lines by exposure to TRAIL, etoposide, HA14-1 and betulinic acid activating the mitochondria by different mechanisms. Sensitivity to TRAIL-induced apoptosis correlated with cleavage of the initiator caspase-8, but the mitochondrial apoptosis pathway was not induced. Similarly, etoposide and betulinic acid could not induce mitochondrial damage. …In contrast, HA14-1 was able to activate mitochondrial apoptosis, thereby demonstrating functionally inducible signalling pathways downstream of the mitochondria. The intactness of the pathways upstream of the mitochondria was shown by pretreatment of TRAIL-sensitive cell lines with HA14-1, which could reconstitute TRAIL-induced mitochondrial damage and resulted in a synergistic apoptosis induction. Our results demonstrate that the apoptotic pathways upstream and downstream of the mitochondria are intact and inducible in renal cell carcinoma cell lines. However, resistance towards mitochondrial apoptosis is located on the level of the mitochondria themselves. Show more

Keywords: Apoptosis, renal cell carcinoma, anticancer drugs, TRAIL, BCL-2

DOI: 10.3233/CLO-2008-0438

Citation: Analytical Cellular Pathology, vol. 30, no. 5, pp. 419-433, 2008

Authors: Kugler, Wilfried | Veenman, Leo | Shandalov, Yulia | Leschiner, Svetlana | Spanier, Ilana | Lakomek, Max | Gavish, Moshe

Article Type: Research Article

Abstract: Background: We have previously shown that the anti-neoplastic agent erucylphosphohomocholine (ErPC3) requires the mitochondrial 18 kDa Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), to induce cell death via the mitochondrial apoptosis pathway. Methods: With the aid of the dye JC-1 and cyclosporin A, applied to glioblastoma cells, we now investigated the significance of opening of the mitochondrial permeability transition pore (MPTP) for ErPC3-induced apoptosis in interaction with the TSPO ligands, PK 11195 and Ro5 4864. Furthermore, we measured cytochrome c release, and caspase-9 and -3 activation in this paradigm. Results: The human glioblastoma cell …lines, U87MG, A172 and U118MG express the MPTP-associated TSPO, voltage-dependent anion channel and adenine nucleotide transporter. Indeed, ErPC3-induced apoptosis was inhibited by the MPTP blocker cyclosporin A and by PK 11195 and Ro5 4864 in a concentration-dependent manner. Furthermore, PK 11195 and Ro5 4864 inhibited collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-9 and -3 activation caused by ErPC3 treatment. Conclusions: This study shows that PK 11195 and Ro5 4864 inhibit the pro-apoptotic function of ErPC3 by blocking its capacity to cause a collapse of the mitochondrial membrane potential. Thus, the TSPO may serve to open the MPTP in response to anti-cancer drugs such as ErPC3. Show more

Keywords: Mitochondrial permeability transition pore, PK 11195, Ro5 4864, TSPO, erucylphosphohomocholine, cancer

DOI: 10.3233/CLO-2008-0431

Citation: Analytical Cellular Pathology, vol. 30, no. 5, pp. 435-450, 2008

Authors: Walen, Kirsten H.

Article Type: Other

DOI: 10.3233/CLO-2008-0444

Citation: Analytical Cellular Pathology, vol. 30, no. 5, pp. 451-452, 2008

Authors: Weaver, Beth A.A. | Silk, Alain D. | Cleveland, Don W.

Article Type: Other

DOI: 10.3233/CLO-2008-0445

Citation: Analytical Cellular Pathology, vol. 30, no. 5, pp. 453-453, 2008

Authors: Bocchetta, Maurizio | Carbone, Michele

Article Type: Other

DOI: 10.3233/CLO-2008-0446

Citation: Analytical Cellular Pathology, vol. 30, no. 5, pp. 455-455, 2008

Authors: Ray, F. Andrew

Article Type: Other

DOI: 10.3233/CLO-2008-0447

Citation: Analytical Cellular Pathology, vol. 30, no. 5, pp. 457-458, 2008