Although photosensitizers, molecules that produce active oxygen species upon activation by visible light, are being extensively used in photodynamic therapy to treat cancer and other clinical conditions, problems include normal cell and tissue damage and associated side effects, which are attributable in part to the fact that cytotoxic effects are largely restricted to the plasma membrane. We have previously shown that the photosensitizer chlorin e 6 has significantly higher photosensitizing activity when present in conjugates containing specific ligands and thus able to be internalized by receptor-expressing cells. In this study we use insulincontaining conjugates to which variants of the simian virus SV40 large tumor antigen nuclear localization signal (NLS) were linked to target chlorin e 6 to the nucleus, a hypersensitive site for active oxygen species-induced damage. NLSs were either included as peptides crosslinked to the carrier bovine serum albumin or encoded within the sequence of a ␤-galactosidase fusion protein carrier. The results for photosensitization demonstrate clearly for the first time that NLSs increase the photosensitizing activity of chlorin e 6 , maximally reducing the EC 50 by a factor of over 2000-fold. This has widereaching implications for achieving efficient cell typespecific photodynamic therapy.Photosensitizers such as porphyrins are molecules that produce active oxygen species upon activation by visible light and are currently being extensively used in photodynamic therapy to treat cancer and other clinical conditions (1-3). Because normal cells are able to accumulate porphyrins, however, and porphyrins are only excreted slowly from the body, prolonged skin photosensitization as well as other effects can be a problem (4, 5), leading to normal cell and tissue damage (6). A high priority with respect to photodynamic therapy is accordingly to increase the specificity of the uptake of photosensitizers in particular target cells, thereby enabling the active dose of porphyrins administered to patients to be reduced. We have previously shown that the photosensitizer chlorin e 6 has significantly higher photosensitizing activity when present in conjugates containing specific ligands such as insulin or concanavalin A and thus is able to be internalized by receptorexpressing cells (7-9). Photosensitization could be competed by incubating cells in the presence of an excess of unconjugated ligand (7-9), indicating that cellular uptake was receptor-dependent. Because only cells expressing specific receptors are targeted in this approach (see Refs. 7-9), it is clear that it enables selectivity in terms of the cell types targeted for photosensitization.Due to the fact that injury induced by singlet oxygen comprising 80% of all of the active oxygen species generated upon porphyrin activation is localized within less than 0.1 m of the site of its production, the effect of photosensitizers is integrally dependent on their site of cellular accumulation (1). Although most porphyrins such as chlorin e 6...

Photosensitizers, molecules that produce active oxygen species upon activation by visible light, are currently being used in photodynamic therapy (PDT) to treat cancer and other conditions, where limitations include normal cells and tissue damage and associated side effects, and the fact that cytotoxic effects are largely restricted to the plasma and other peripheral membranes. In this study, we used insulincontaining conjugates to which variants of the simian-virus-SV40 large-tumor antigen (T-ag) nuclear localization signal (NLS) were linked in order to target the photosensitizer chlorin e 6 to the nucleus. NLSs were included either as peptides coupled co-valently to the carrier bovine serum albumin, or within the coding sequence of ␤-galactosidase fusion proteins. The most potent photosensitizing conjugate was the NLS-containing T-ag ␤-galactosidase fusion protein (P10)-(chlorin e 6 )-insulin, exhibiting an EC 50 more than 2400-fold lower than the value for free chlorin e 6 , and more than 15-fold lower than that of an NLS-deficient ␤-galactosidase-(chlorin e 6 )-insulin construct, thus demonstrating that NLSs can increase the photosensitizing activity of chlorin e 6 . Attenuated adenoviruses were used to increase the nuclear delivery of conjugates through its endosomal-membrane-disrupting activity. In the case of the NLS-containing P10-conjugate, co-incubation with adenovirus increased the proportion of cells whose nuclear photosensitizing activity was higher than that in the cytoplasm by 2.5-fold. This use of adenoviruses in conjunction with photosensitizers has clear implications for achieving efficient cell-type-specific PDT. Int. J. Cancer 81:734-740, 1999.1999 Wiley-Liss, Inc.

Experiments with human hepatoma PLC/PRF/5 cells and human embryo skin fibroblasts involving the use of three different tests (colony formation, Trypan blue exclusion, labeled thymidine incorporation) have demonstrated a significantly higher photosensitizing activity of chlorin e6 conjugates with internalizable ligands as compared to that of chlorin e6 itself. Receptor-mediated internalization of chlorin e6 conjugates ensures a greater photosensitization of cells than binding of those conjugates to cell surface receptors. The suitability of such conjugates that permit the delivery of a photosensitizer to sensitive intracellular targets is discussed.

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Photosensitizers, molecules that produce active oxygen species upon activation by visible light, are currently being used in photodynamic therapy (PDT) to treat cancer and other conditions, where limitations include normal cells and tissue damage and associated side effects, and the fact that cytotoxic effects are largely restricted to the plasma and other peripheral membranes. In this study, we used insulin‐containing conjugates to which variants of the simian‐virus‐SV40 large‐tumor antigen (T‐ag) nuclear localization signal (NLS) were linked in order to target the photosensitizer chlorin e6 to the nucleus. NLSs were included either as peptides coupled co‐valently to the carrier bovine serum albumin, or within the coding sequence of β‐galactosidase fusion proteins. The most potent photosensitizing conjugate was the NLS‐containing T‐ag β‐galactosidase fusion protein (P10)‐(chlorin e6)‐insulin, exhibiting an EC50 more than 2400‐fold lower than the value for free chlorin e6, and more than 15‐fold lower than that of an NLS‐deficient β‐galactosidase‐(chlorin e6)‐insulin construct, thus demonstrating that NLSs can increase the photosensitizing activity of chlorin e6. Attenuated adenoviruses were used to increase the nuclear delivery of conjugates through its endosomal‐membrane‐disrupting activity. In the case of the NLS‐containing P10‐conjugate, co‐incubation with adenovirus increased the proportion of cells whose nuclear photosensitizing activity was higher than that in the cytoplasm by 2.5‐fold. This use of adenoviruses in conjunction with photosensitizers has clear implications for achieving efficient cell‐type‐specific PDT. Int. J. Cancer 81:734–740, 1999. © 1999 Wiley‐Liss, Inc.

Cell regulation of Ph+cell proliferation and differentiation has been studied ex vivo in various chronic myeloid leukemia (CML) patients. The regulation is provided by alternation of effective stages of proliferation and maturation with inhibition of Ph+ cell proliferation by accumulating neutrophils under apoptosis blockage. The alternation of stages consists of switching stage 1 (effective proliferation) to stage 2 (effective maturation) and proceeds according to the 1/2 -1/2/1 or 2/1-2/1/2/1 schemes. The kinetic plots of alternations pass through control points of crossing plots, where the parameters of proliferation and maturation are equal. The indices of P/D efficiency (ratio of proliferation and maturation rates) are 1.06±0.23 and don't depend on time, alternation order, or sources of Ph+ cells - CML patients. During stages alternation, conversely, the parameters of Ph+ cell proliferation and maturation vary. The proliferation stages are characterized by increased proliferating cells content, a decreased number of neutrophils, and apoptosis induction. At the maturation stages, conversely, apoptosis is inhibited, the number of mature neutrophils increases, while immature Ph+ cells decrease. High content neutrophils inhibit the proliferation of Ph+ cells and impair their own maturation by inversion of maturation order, probably through a feedback mechanism. The regulation differences ex vivo reveal three types of Ph+ cells from various individual CML patients, distinguished by the number and duration of alternating stages of proliferation and maturation. Ph+ cells types 1 and 2 have one prolonged stage of effective proliferation or effective maturation with efficiency indices P/D1 = 1-20 or P/D2 ⇐ 1. At the same time period, the proliferation and differentiation of the Ph+ cells type 3 proceeds with repeated alternations of stages with P/D1 = 1-4 or P/D2 ⇐ 1. Type 1 Ph+ cells (~20%) were isolated from patients in advanced stages of CML, while Ph+ cells types 2 and 3 (30 and 50% correspondingly) were isolated from CML chronic phase patients sensitive to chemotherapy.

Tumor antigens recognized by CTLs have been identified several years ago and are major targets for creating anticancer vaccines. PRAME is an antigen which is highly expressed in various malignant tumors including melanomas and hematopoietic malignancies such as acute and chronic leukemias (AML, CML). Technology for producing recombinant antigen PRAME is based on creating a bacterial producer strain containing cDNA of human PRAME gene. We have obtained two producers of recombinant PRAME protein and its N-half, the synthesis of the target protein in the producers occurs in the inclusion bodies. The schemes of isolation and purification of soluble proteins have been developed. The protein purity was approximately 95-96%. The monoclonal antibodies raised against truncated recombinant PRAME were used for PRAME protein analysis by Western blot on the various tumor cells. Specific monoclonal antibodies recognized the native PRAME protein in tumor cell lines as well as in tumor samples from patients. Our findings support the suggestion that this recombinant antigen may be further used as a target for diagnostic and therapeutic approaches. The monoclonal antibodies can be used for immunoassays of tumor samples from patients with hematologic malignancies to reveal clinical features and to monitor tumor progression.

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