Eradication of Breast Cancer Xenografts by Hyperthermic Suicide Gene Therapy under the Control of the Heat Shock Protein Promoter

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Abstract

To investigate the usefulness of heat shock protein (HSP) promoter for breast cancer gene therapy, hyperthermia and HSV thymidine kinase (tk) suicide gene combination therapy was examined with mouse mammary cancer cell line FM3A. HSP promoter activity was markedly increased after heat shock (41-45 degrees C), with maximum activation (about 400-fold) at 3 hr. An in vitro cytotoxic assay showed that HSP-tk-transduced FM3A cells became more sensitive (more than 50,000 times) to ganciclovir (GCV) with heat shock, but untreated cells showed no increased cytotoxic sensitivity to GCV compared with control FM3A cells. In addition to promoter-oriented selective cell killing, a "chemosensitization effect" as a bystander effect was demonstrated by hyperthermia and suicide gene combination therapy, using a non-heat-inducible promoter. Immunohistochemical analysis revealed that this synergistic killing effect was dependent on apoptotic cell death with upregulation of both Fas and FasL (Fas ligand) expression. We also examined the efficacy of HSP-tk gene therapy in vivo by implanting breast cancer in subcutaneous and intraperitoneal models of BALB/c nude mice targeted by the HVJ-anionic liposome method. Significant tumor regression was observed in HSP-tk-transduced tumors followed by hyperthermia therapy, but no such inhibition was noted in either the mock vector transfection or hyperthermia group compared with control tumor-bearing mice. Our results demonstrate that this combination system is synergistically effective in mediating Fas-dependent apoptosis for a specific gene therapy targeting HSP-expressing mammary carcinomas, even in advanced and heat-resistant breast cancer.

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Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation

SA Ben-Sasson, Y Sherman, Y Gavrieli (1992)

Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy- transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.

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Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis.

David Caron, Allan Massie, Maria C Denis … (1997)

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.

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VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer.

Reija Valtola, Petri Salven, Päivi Heikkilä … (1999)

Recently, monoclonal antibodies against the human vascular endothelial growth factor receptor VEGFR-3 were shown to provide a specific antigenic marker for lymphatic endothelium in various normal tissues. In this study we have investigated the expression of VEGFR-3 and its ligand VEGF-C in normal breast tissue and in breast tumors by immunohistochemistry. VEGFR-3 was weakly expressed in capillaries of normal breast tissue and in fibroadenomas. In intraductal breast carcinomas, VEGFR-3 was prominent in the "necklace" vessels adjacent to the basal lamina of the tumor-filled ducts. VEGF receptor 1 and 2 as well as blood vessel endothelial and basal lamina markers were colocalized with VEGFR-3 in many of these vessels. Antibodies against smooth muscle alpha-actin gave a weak staining of the necklace vessels, suggesting that they were incompletely covered by pericytes/smooth muscle cells. A highly elevated number of VEGFR-3 positive vessels was found in invasive breast cancer in comparison with histologically normal breast tissue (P < 0.0001, the Mann-Whitney test). VEGF-C was located in the cytoplasm of intraductal and invasive cancer cells. The results demonstrate that the expression of VEGFR-3 becomes up-regulated in the endothelium of angiogenic blood vessels in breast cancer. The results also suggest that VEGF-C secreted by the intraductal carcinoma cells acts predominantly as an angiogenic growth factor for blood vessels, although this paracrine signaling network between the cancer cells and the endothelium may also be involved in modifying the permeabilities of both blood and lymphatic vessels and metastasis formation.