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Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. These results provide a proof of principle that PIKfyve specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro., (Copyright © 2021. Published by Elsevier Inc.)

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC 50 at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.

LSD1/KDM1 is a histone demethylase that preferentially removes methyl groups from the mono- and di-methylated lysine 4 in histone H3 (H3K4), key marks for active chromatin for transcriptional activation. LSD1 is essential for pluripotent embryonic stem cells and embryonic teratocarcinoma/carcinoma cells and its expression is often elevated in various cancers. We developed a new LSD1 inhibitor, CBB3001, which potently inhibited LSD1 activity both in vitro and in vivo. CBB3001 also selectively inhibited the growth of human ovarian teratocarcinoma PA-1 and mouse embryonic carcinoma F9 cells, caused the downregulation of pluripotent stem cell proteins SOX2 and OCT4. However, CBB3001 does not have significant inhibition on the growth of human colorectal carcinoma HCT116 cells or mouse fibroblast NIH3T3 cells that do not express these stem cell proteins. Our studies strongly indicate that CBB3001 is a specific LSD1 inhibitor that selectively inhibits teratocarcinoma and embryonic carcinoma cells that express SOX2 and OCT4., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Gedunin is one of the major compounds found in the neem tree (Azadirachta indica) . In the present study, antiproliferative potential of gedunin was evaluated in human embryonal carcinoma cells (NTERA-2, a cancer stem cell model) and peripheral blood mononuclear cells (PBMCs), using Sulforhodamine (SRB) and WST-1 assays, respectively. The effects of gedunin on expression of heat shock protein 90 (HSP90), its cochaperone Cdc37, and HSP client proteins (AKT, ErbB2, and HSF1) were evaluated by real-time PCR. Effects of gedunin on apoptosis were evaluated by (a) apoptosis associated morphological changes, (b) caspase 3/7 expression, (c) DNA fragmentation, (d) TUNEL assay, and (e) real-time PCR of apoptosis related genes ( Bax , p53, and survivin ). Gedunin showed a promising antiproliferative effect in NTERA-2 cells with IC 50 values of 14.59, 8.49, and 6.55  μ g/mL at 24, 48, and 72 h after incubations, respectively, while exerting a minimal effect on PBMCs. Expression of HSP90, its client proteins, and survivin was inhibited and Bax and p53 were upregulated by gedunin. Apoptosis related morphological changes, DNA fragmentation, and increased caspase 3/7 activities confirmed the proapoptotic effects of gedunin. Collectively, results indicate that gedunin may be a good drug lead for treatment of chemo and radiotherapy resistant cancer stem cells.

This study aims to unravel the use of GSK-3 inhibitors as viable apoptotic inducers for teratocarcinoma-derived ovarian PA-1 cells. MTT assay was carried out to assess inhibitory concentrations of LiCl and TDG. AO/EB staining and Hoechst 33258 staining were employed to assess the damage. Mitochondrial membrane potential (ΔΨm) and ROS generation were assessed with IC50 concentrations of LiCl and TDG. Tumor-related genes (p53, p21, IL-8, TNF-α, MMP-2, Fas-L, Cox-2, and caspase-3) were assessed with 1/4 IC50, 1/2 IC50, IC50 concentrations by semi-quantitative RT- PCR. Cell cycle analysis was performed with IC50 concentration of LiCl and TDG. Western blot analysis was performed for caspase-3, caspase-7, caspase-9, PARP to estimate the possible damage induced by GSK-3 inhibitors and regulation of GSK-3β, pGSK-3β, Cox-2. GSK-3 inhibitors demonstrated a concentration and time-dependent reduction in cell viability, exhibiting significant ROS generation and reduced ΔΨm at their IC50 values. Substantial concentration-dependent gene expression changes with significant upregulation of P21, Cox-2, TNF-α, caspase-3, Fas-L were observed. Protein expression of caspase-3 caspase-7, caspase-9, PARP exhibited significant cleavage in LiCl and TDG-treated cells. Protein expression of Cox-2 was significantly increased in IC50 concentration of TDG. Cell cycle analysis showed significant accumulation of cells at sub-G0-G1.

Background: Deficiency in Vitamin D3 (cholecalciferol) may predispose to some malignancies, including gonadal tumors and in experimental models vitamin D3 has been proven to inhibit the growth of cancer cells. To learn more about the potential role of vitamin D3 in cancerogenesis, we evaluated the expression and functionality of the vitamin D receptor (VDR) and its role in metastasis of ovarian cancer cells and of murine and human teratocarcinoma cell lines., Methods: In our studies we employed murine embrynic stem cells (ESD3), murine (P19) and human (NTERA-2) teratocarcimona cells lines, human ovarian cancer cells (A2780) as well as purified murine and human purified very small embryonic like stem cells (VSELs). We evaluated expression of Vitamin D3 receptor (VDR) in these cells as well as effect of vitamin D3 exposure on cell proliferation and migration., Results: We here provide also more evidence for the role of vitamin D3 in germline-derived malignancies, and this evidence supports the proposal that vitamin D3 treatment inhibits growth and metastatic potential of several germline-derived malignancies. We also found that the ESD3 murine immortalized embryonic stem cell line and normal, pluripotent, germline-marker-positive very small embryonic-like stem cells (VSELs) isolated from adult tissues are stimulated by vitamin D3, which suggests that vitamin D3 affects the earliest stages of embryogenesis., Conclusions: We found that however all normal and malignant germ-line derived cells express functional VDR, Vitamin D3 differently affects their proliferation and migration. We postulate that while Vitamin D3 as anticancer drug inhibits proliferation of malignant cells, it may protect normal stem cells that play an important role in development and tissue/organ regeneration.

We describe the expression and in vivo characterization of an antibody-cytokine fusion protein, based on murine Interleukin-13 (IL13) and the monoclonal antibody F8, specific to the alternatively spliced extra domain A of fibronectin, a marker of neo-angiogenesis. The IL13 moiety was fused at the C-terminal extremity of the F8 antibody in diabody format. The resulting F8-IL13 immunocytokine retained the full binding properties of the parental antibody and cytokine bioactivity. The fusion protein could be expressed in mammalian cells, purified to homogeneity and showed a preferential accumulation at the tumor site. When used as single agent at doses of 200 μg, F8-IL13 exhibited a strong inhibition of tumor growth rate in two models of cancer (F9 teratocarcinoma and Wehi-164), promoting an infiltration of various types of leukocytes into the neoplastic mass. This anticancer activity could be potentiated by combination with an immunocytokine based on the F8 antibody and murine IL12, leading to complete and long-lasting tumor eradications. Mice cured from Wehi-164 sarcomas acquired a durable protective antitumor immunity, and selective depletion of immune cells revealed that the antitumor activity was mainly mediated by cluster of differentiation 4-positive T cells. This study indicates that IL13 can be efficiently delivered to the tumor neo-vasculature and that it mediates a potent anticancer activity in the two models of cancer investigated in this study. The observed mechanism of action for F8-IL13 was surprising, since immunocytokines based on other payloads (e.g., IL2, IL4, IL12 and TNF) eradicate cancer by the combined contribution of natural killer cells and cluster of differentiation 8-positive T cells.

There is an increasing biotechnological interest in 'arming' therapeutic antibodies with bioactive payloads. Many antibody-cytokine fusion proteins (immunocytokines) have been described and some of these biopharmaceuticals have progressed to clinical studies. Here, we describe for the first time the expression and in vivo characterization of immunocytokines based on murine IL1β and IL6. These potent pro-inflammatory cytokines were fused at the N-terminus or at the C-terminus of the monoclonal antibodies F8 (specific to the alternatively-spliced extra-domain A domain of fibronectin, a marker of tumor angiogenesis). All immunocytokines retained the binding properties of the parental antibody and were homogenous, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, except for the N-terminal fusion of IL1β which revealed the presence of glycosylated species. When analyzed by quantitative biodistribution analysis using radioiodinated protein preparations, F8 fusions with IL6 revealed a preferential accumulation at the tumor site for both cytokine orientations, whereas IL1β fusions exhibited lower tumor to organ ratios and a slower blood clearance profile. The fusion proteins with the cytokine payload at the C-terminus were studied in therapy experiments in immunocompetent mice bearing F9 tumors. Immunocytokines based on IL1β resulted in 10% body weight loss at a 5-µg dose, whereas IL6-based products caused a 5% body weight loss at a 225-µg dose. Both F8-IL1β and F8-IL6 exhibited a <50% inhibition of tumor growth rate, which was substantially lower than the one previously reported for F8-TNF, a closely related pro-inflammatory immunocytokine. This study indicates that IL6 can be efficiently delivered to the tumor neo-vasculature by fusion with the F8 antibody. While F8-IL6 was not as potent as other F8-based immunocytokines that exhibit similar biodistribution profiles, the fusion protein sheds light on the different roles of pro-inflammatory cytokines in boosting immunity against the tumor., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Object: Antiangiogenic treatments are beginning to give promising outcomes in many vascular diseases including tumor angiogenesis. In this current study the antiangiogenic and pro-apoptotic actions of α1(IV)NC1 and its N- and C- peptides α1S1(IV)NC1, α1S2(IV)NC1 were investigated in-vitro and in-vivo., Study Method: Endothelial cells (ECs) were treated with α1(IV)NC1, α1S1(IV)NC1, α1S2(IV)NC1 and in-vitro proliferation, migration, tube formation and apoptotic assays were executed. FasL, Fas, Caspase-8, -3 and PARP activations were studied using immunoblotting analysis using specific antibodies. Also the in-vivo antiangiogenic and pro-apoptotic effects were tested using α1(IV)NC1 in a mice model., Results: Like α1(IV)NC1, its N- and C- terminal α1S2(IV)NC1 and α1S1(IV)NC1 domains posses anti-proliferative, pro-apoptotic activity and inhibit ECs migration and tube formation in-vitro. Both α1S1(IV)NC1 and α1S2(IV)NC1 domains promote apoptosis by activating FasL and down stream apoptotic events including activation of caspase-8, -3 and PARP cleavage in a dose dependent manner in-vitro in ECs. Tumors in mice showed apoptotic TUNEL positive microvasculature upon α1(IV)NC1 treatment, indicating inhibition of tumor angiogenesis and tumor growth. Further, the antitumor activity of α1(IV)NC1 was abrogated when caspase-3 inhibitor was used. These results conform additional properties of α1(IV)NC1 as an endogenous angioinhibitor that induces apoptosis in-vitro and in-vivo by activating FasL mediated caspase-3., Significance: α1(IV)NC1 and its N- and C- terminal α1S1(IV)NC1 and α1S2(IV)NC1 domains also posses pro-apoptotic and angioinhibitory activity in-vitro and in-vivo. α1(IV)NC1 regulates tumor angiogenesis by activating FasL mediated apoptosis in-vitro and in-vivo. These results demonstrate that α1(IV)NC1 and its peptides inhibit neo-vascular diseases.

Fatty acid-binding protein 3 (FABP3) is a low molecular weight protein with distinct tissue distribution, which may play an important role in fatty acid transport, cell growth, cellular signaling, and gene transcription. We have previously shown FABP3 was more highly expressed in myocardium with ventricular septal defects than in normal myocardium and furthermore, that overexpression of FABP3 causes mitochondrial dysfunction and induces apoptosis in the P19 mouse teratocarcinoma cell line (P19), which is a suitable model for the investigation of cardiac differentiation at the molecular and functional levels. α-Lipoic acid (α-LA), a natural dithiol compound with antioxidant properties, has been reported to protect mitochondrial function in cells. In this study, we established an FABP3-overexpressing P19 cell line for the investigation of the impact of α-LA on mitochondrial impairment and apoptosis in these cells. Mitochondrial morphology was evaluated by transmission electron microscopy, while the effects of α-LA on reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), intracellular ATP content and the amount of mitochondrial DNA were analyzed by flow cytometry, a commercially available assay and quantitative real-time PCR, respectively. The results revealed that α-LA ameliorated mitochondrial deformation and decreased intracellular ROS production. Furthermore, the MMP, intracellular ATP synthesis and the amount of mitochondrial DNA were also increased. Most significantly, α-LA was shown to reverse apoptosis. Collectively, our results indicate that abnormalities in FABP3 expression contribute to mitochondrial dysfunction and apoptosis, and that α-LA represents a suitable candidate for development as a treatment for apoptosis-related congenital cardiac malformations.

In this work, we have analyzed the expression of different members of the ErbB family in human samples of testicular germ cell tumors (GCTs). We observed expression of ErbB1 or ErbB2 in different tumor subtypes, but we also found high expression of ErbB3 in all GCTs tested. This pattern of expression was maintained when primary tumors were orthotopically implanted in nude mice. We have chosen a choriocarcinoma model characterized by high levels of ErbB1, but also of ErbB2 and ErbB3, to assay the in vivo effect of ErbB inhibitors on tumoral growth. Our results showed a complete lack of effect (refractoriness) to the pure ErbB1 receptor inhibitors cetuximab and gefitinib. While these inhibitors blocked ErbB1 phosphorylation, ErbB2 phosphorylation was not affected, suggesting an ErbB1-independent activation of this receptor. To confirm the importance of ErbB2 activation, animals were treated with lapatinib, a dual ErbB1 and ErbB2 inhibitor. Lapatinib treatment caused a 50% inhibition in tumor growth, an effect correlated with a blockade of both ErbB1 and ErbB2 phosphorylation levels, and of downstream signaling pathways (Akt, ERKs and Stat3). ErbB2 activation could still occur due to the formation of ErbB2/ErbB3 heterodimers, and ErbB3 activation was completely inhibited by lapatinib. Finally, combined inhibition of ErbB1 (gefitinib) and ErbB3 activities (knockdown expression by shRNA) inhibited tumoral testicular cells proliferation in a similar way to lapatinib. Our results explain why lapatinib but not anti-ErbB1 agents might be effective for treatment of testicular GCT patients., (Copyright © 2013 UICC.)

Systemic high-dose IL2 promotes long-term survival in a subset of metastatic melanoma patients, but this treatment is accompanied by severe toxicities. The immunocytokine L19-IL2, in which IL2 is fused to the human L19 antibody capable of selective accumulation on tumor neovasculature, has recently shown encouraging clinical activity in patients with metastatic melanoma. In this study, we have investigated the therapeutic performance of L19-IL2, administered systemically in combination with a murine anti-CTLA-4 antibody or with a second clinical-stage immunocytokine (L19-TNF) in two syngeneic immunocompetent mouse models of cancer. We observed complete tumor eradications when L19-IL2 was used in combination with CTLA-4 blockade. Interestingly, mice cured from F9 tumors developed new lesions when rechallenged with tumor cells after therapy, whereas mice cured from CT26 tumors were resistant to tumor rechallenge. Similarly, L19-IL2 induced complete remissions when administered in a single intratumoral injection in combination with L19-TNF, whereas the two components did not lead to cures when administered as single agents. These findings provide a rationale for combination trials in melanoma, as the individual therapeutic agents have been extensively studied in clinical trials, and the antigen recognized by the L19 antibody has an identical sequence in mouse and man.

Although biological therapy has shown promising clinical responses in many cancers including metastatic melanoma, only a subset of patients has shown marked regression of lesions. In most patients, systemic administration of biological therapies with cytokines is associated with severe toxicities. Schwager et al., in this issue of Journal of Investigative Dermatology, have examined the role of immunocytokines L19-IL2 and L19-TNF to minimize toxicities, and in combination with Ipilimumab they report complete regression of tumors using syngeneic mouse models. The results, if confirmed in clinical trials, will have major implications for the treatment of human cancers, including melanomas.

Using 22R-hydroxycholesterol as a sub-structure to screen natural compound databases, we identified a naturally occurring steroid (sc-7) with a 16-acetoxy-22R-hydroxycholesterol moiety, in which the hydroxyl groups in positions 3 and 22 are esterified by an acetoxy group and in which the carbon in position 26 carries a functional diacetylamino. sc-7 is an analog of the sex steroids dehydro-oogoniol and antheridiol, can be isolated from the water mold Achlya heterosexualis, and promoted neurogenesis in vitro and in vivo. Mouse embryonic teratocarcinoma P19 cells exposed to sc-7 for 2days followed by a 5-day wash-out differentiated into cholinergic neurons that expressed specific neuronal markers and displayed axonal formation. Axons continued growing up to 28days after treatment. In vivo, infusion of sc-7 for 2weeks into the left ventricle of the rat brain followed by a 3-week wash-out induced bromodeoxyuridine uptake by cells of the ependymal layer and subventricular zone that co-localized with doublecortin and glial fibrillary acidic protein immunostaining, demonstrating induction of proliferation and differentiation of neuronal progenitors. Migrating neuroblasts were also observed in the corpus callosum. Thus, under these experimental conditions, adult ependymal cells resumed proliferation and differentiation. Taken together, these results suggest that sc-7 is an interesting molecule for stimulating in situ neurogenesis from resident neuronal progenitors as part of neuron replacement therapy. sc-7 did not bind to nuclear steroid receptors and was not metabolized as a steroid, supporting our hypothesis that the neurogenic effect of sc-7 is not likely due to a steroid-like effect., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The antibody-mediated targeted delivery of cytokines, growth factors and immunomodulators offers great potential for the therapy of cancer and other serious conditions. Interferon-alpha has long been used in the clinic for the treatment of patients with certain malignancies or with viral disease. Promising anticancer activity has recently been reported for two fusion proteins consisting of immunoglobulins bearing the interferon-alpha polypeptide at the C-terminal end of the molecule. Here we describe the design, production and characterization of a novel immunocytokine, in which murine interferon-alpha2 was sequentially fused with the tumor-targeting antibody fragment scFv(F8), specific to the alternatively-spliced EDA domain of fibronectin. The resulting fusion protein (F8-IFNa) could be produced to homogeneity and was shown to retain both antigen binding activity and interferon-alpha activity. Biodistribution studies in tumor-bearing mice with radioiodinated protein preparations confirmed the ability of F8-IFNa to selectively localize at the tumor site. However, using two different murine models of cancer (F9 teratocarcinomas and Cloudman S91 melanomas in immunocompetent mice), we could not detect a striking superiority for the therapeutic performance of F8-IFNa as compared to KSF-IFNa, a fusion protein of irrelevant specificity in the mouse which was used as negative control. In the paper, we present hypotheses why the antibody-based pharmacodelivery of interferon-alpha fails to eradicate tumors, in contrast to the situation observed by our group for other immunocytokines, which benefit from a selective localization at the tumor site.

Unlabelled: Cisplatin (CDDP) and oxaliplatin (OXA-P) are potential therapeutic drugs in the treatment of testicular cancer. However, the emergence of drug resistance has been documented not only in patients after chemotherapy but also subsequently to fractionated X-irradiation. Specific radiation-induced biochemical alterations may play a role in the observed resistance. Since irradiation influences the cellular responses to chemotherapy, this study investigated changes in the expression of key proteins in the regulation of DNA repair and apoptosis subsequent to sequential irradiation., Materials and Methods: Logarithmically growing human teratocarcinoma cell lines 2101 EP and H 12.1 were irradiated (10 fractions of 4 Gy in vitro) to establish the sub-lines 2101 EP/DXR-10 and H 12.1/DXR-10. Radiosensitivity was assayed using a clonogenic survival assay. Drug response was assayed using a sulforhodamine B assay. Expression of p53, PARP, hMSH2 and Fas was detected by Western blotting., Results: Both DXR-10 sub-lines showed a significant increase in sensitivity towards CDDP as compared to their parental cell line, however, there was a concomitant increase in resistance against OXA-P. No significant changes in radiosensitivity between parental and DXR-10 cell lines were observed. In addition, there was an up-regulation of PARP, p53, hMSH2 and Fas in the DXR-10 sub-lines, implicating induced damage tolerance and repair mechanisms following irradiation., Conclusion: These results suggest that radiation preceding chemotherapy might induce resistance to subsequent chemotherapy with oxaliplatin but not to cisplatin. This is a novel observation and, if confirmed, particularly in other tumor types, may have clinical implications.

In our previous study, PAb, a VEGF polyclonal antibody was found to inhibit murine tumor growth significantly. The main objective of this study was to investigate the efficacy of combination therapy of (PAb) and cisplatin on human ovarian cancer xenograft. Effect of VEGF, PAb, PAb-cisplatin combination, and cisplatin alone on cultured human ovarian teratocarci-noma cell line PA-1 were assessed by measuring cell proliferation, matrigel invasion, MMP-9 expression, and MMP-9 secretion. In vivo, effect of PAb was observed in a xenograft model of ovarian cancer. Antitumor efficacy was monitored by assessment of tumor volume, MVD, serum NO, serum VEGF, and p53 expression. VEGF increased proliferation of PA-1 cell in a dose-dependent manner while addition of PAb inhibited cell proliferation, cell invasion as well as MMP-9 secretion in vitro. Tumor burden in PAb and PAb-cisplatin combination group was reduced by 41% (p < 0.05) and 66% (p < 0.01), respectively. A significant decrease in MVD, serum NO, serum VEGF, and p53 expression was also observed after PAb and PAb-cisplatin combination treatment when compared to normal mouse serum IgG-treated control mice. Thus, it was concluded that VEGF immunoneutralization may enhance cisplatin-in-duced apoptosis in human ovarian cancer and thus may be an effective way to reduce tumor growth in ovarian carcinoma.

Aims and Background: Analysis of patients with late relapse of testicular germ cell tumors (GCTs) with reports on clinicopathological features and outcomes., Methods: We identified all patients diagnosed with testicular GCTs at our Institute between 1988 and 2004 who developed relapse > or = 24 months after completion of primary therapy. A retrospective case-note review was performed to extract clinical, pathological, treatment and outcome data., Results: Six patients (1.25%) developed late relapse. All patients presented with stage I disease and were classified as "good risk" according to the International Germ Cell Consensus Classification. Mean time to late relapse was 48 months. Markers at late relapse were normal in all patients. Relapse was confined to retroperitoneal sites in five patients and located in the mediastinum in one patient. Five patients were managed by chemotherapy alone while one underwent combined treatment with surgery followed by chemotherapy. All patients obtained a complete response and all remained free from recurrence with a mean follow-up of 115 months., Conclusions: The incidence of late relapse in this small series is low. Chemonaive patients with late relapse were successfully salvaged with chemotherapy alone or surgical excision followed by cisplatin-based chemotherapy. The optimal duration of follow-up in patients with testicular GCTs is not known and practice varies widely. At our Institute we advise lifelong follow-up of all patients with malignant GCTs of the testis.

Human alpha3 chain, a noncollagenous domain of type IV collagen [alpha3(IV)NC1], inhibits angiogenesis and tumor growth. These biologic functions are partly attributed to the binding of alpha3(IV)NC1 to alphaVbeta3 and alpha3beta1 integrins. alpha3(IV)NC1 binds alphaVbeta3 integrin, leading to translation inhibition by inhibiting focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathways. In the present study, we evaluated the role of alpha3beta1 and alphaVbeta3 integrins in tube formation and regulation of cyclooxygenase-2 (COX-2) on alpha3(IV)NC1 stimulation. We found that although both integrins were required for the inhibition of tube formation by alpha3(IV)NC1 in endothelial cells, only alpha3beta1 integrin was sufficient to regulate COX-2 in hypoxic endothelial cells. We show that binding of alpha3(IV)NC1 to alpha3beta1 integrin leads to inhibition of COX-2-mediated pro-angiogenic factors, vascular endothelial growth factor, and basic fibroblast growth factor by regulating IkappaBalpha/NFkappaB axis, and is independent of alphaVbeta3 integrin. Furthermore, beta3 integrin-null endothelial cells, when treated with alpha3(IV)NC1, inhibited hypoxia-mediated COX-2 expression, whereas COX-2 inhibition was not observed in alpha3 integrin-null endothelial cells, indicating that regulation of COX-2 by alpha3(IV)NC1 is mediated by integrin alpha3beta1. Our in vitro and in vivo findings demonstrate that alpha3beta1 integrin is critical for alpha3(IV)NC1-mediated inhibition of COX-2-dependent angiogenic signaling and inhibition of tumor progression.

Wnt signaling is important for normal cell proliferation and differentiation, and mutations in pathway components are associated with human cancers. Recent studies suggest that altered wnt ligand/receptor interactions might also contribute to human tumorigenesis. Therefore, agents that antagonize wnt signaling at the extracellular level would be attractive therapeutics for these cancers. We have generated a soluble wnt receptor comprising the Frizzled8 cysteine-rich domain (CRD) fused to the human Fc domain (F8CRDhFc) that exhibits favorable pharmacologic properties in vivo. Potent antitumor efficacy was shown using the mouse mammary tumor virus-Wnt1 tumor model under dosing conditions that did not produce detectable toxicity in regenerating tissue compartments. In vitro, F8CRDhFc inhibited autocrine wnt signaling in the teratoma cell lines PA-1, NTera-2, Tera-2, and NCCIT. In vivo, systemic administration of F8CRDhFc significantly retarded the growth of tumor xenografts derived from two of these cell lines, PA-1 and NTera-2. Pharmacodynamic markers of wnt signaling, identified by gene expression analysis of cultured teratoma cells, were also modulated in the tumor xenografts following treatment with F8CRDhFc. Additionally, these markers could be used as indicators of treatment efficacy and might also be useful in identifying patients that would benefit from the therapeutic agent. This is the first report showing the efficacy of a soluble wnt receptor as an antitumor agent and suggests that further development of wnt antagonists will have utility in treating human cancer.

First line treatment of patients pts with poor-prognosis GCT, using BEP, is unsatisfactory. T-BEP (paclitaxel followed by BEP) demonstrated promising efficacy in the group of pts with intermediate and poor prognosis GCT. We present the results achieved with 1st line T-BEP in pts with poor-prognosis CGT. Twenty-four pts received T-BEP as initial therapy. Three pts (12.5%) had primary mediastinal GCT. Four cycles of T-BEP were given 21 days apart. Paclitaxel 175 mg/m2 was administered on day 1 before administration of BEP. The administration of G-CSF was not scheduled. Surgical resection of all radiographic residua was considered. All pts were assessable for response. Complete or partial response with negative tumor markers was achieved in 13 pts (54.2%; CI 95%: 34.3-74.1%). Median follow-up is 35.6 months. Median survival was not achieved and median time-to-progression is 9.5 months. Myelosuppression was the major toxicity with Gr3-4 granulocytopenia experienced in 52.1% of all courses. There were two treatment-related deaths due to sepsis. Patients treated with 1st line T-BEP didn't achieve higher response rate or time to progression. However, the overall survival observed in our study is surprisingly long. We do not recommend using this regimen without G-CSF support due to substantial toxicity.

The irregular vasculature and high interstitial pressure of solid tumors hinder the delivery of cytotoxic agents to cancer cells. As a consequence, the doses of chemotherapy necessary to achieve complete tumor eradication are associated with unacceptably high toxicities. The selective thrombosis of tumor blood vessels has been postulated as an alternative avenue for combating cancer, depriving tumors of nutrients and oxygen and causing an avalanche of tumor cell deaths. The human antibody L19, specific to the EDB domain of fibronectin, a marker of angiogenesis, is capable of selective in vivo localization around tumor blood vessels and is thus a suitable agent for delivering toxic payloads to the tumor neovasculature. Here we show that a chemical conjugate of the L19 antibody with the photosensitizer bis(triethanolamine)Sn(IV) chlorin e(6), after intravenous injection and irradiation with red light, caused an arrest of tumor growth in mice with subcutaneous tumors. By contrast, a photosensitizer conjugate obtained with an antibody of identical pharmacokinetic properties but irrelevant specificity did not exhibit a significant therapeutic effect. These results confirm that vascular targeting strategies, aimed at the selective occlusion/disruption of tumor blood vessels, have a significant anticancer therapeutic potential and encourage the use of antibody-photosensitizer conjugates for the therapy of superficial tumors and possibly other angiogenesis-related pathologies.

The murine embryonal teratocarcinoma cell line, P19, was genetically manipulated in order to provide preliminary information on compounds that induce differentiation. Without chemical induction, P19 cells remain in an undifferentiated state, but can be induced to differentiate into specific cell types. For example, dimethyl sulphoxide (DMSO) induces cardiac and skeletal muscle differentiation, whereas retinoic acid stimulates neuronal differentiation. P19 cells were transfected with a construct containing a segment of the murineTert (mTert) promoter sequence combined with the green fluorescent protein (GFP) gene, which acts as a reporter gene. mTert expression, the reverse transcriptase component of murine telomerase, is closely linked to telomerase activity and is down-regulated during differentiation. Three retinoids and DMSO induced the differentiation of P19 cells, which was determined by a reduction in mTert&#95;GFP expression, detected by flow cytometry and confocal microscopy as independent methods of detection. A test substance, ethanol, and a control substance, saccharin, did not cause a decrease in mTert&#95;GFP expression. In addition, it could be demonstrated that the mTert&#95;GFP test detects developmentally relevant effects at non-cytotoxic concentrations. The ID50 values derived for the reduction of mTert&#95;GFP expression were lower than the IC50 values detected with the MTT test, by a factor of 21.4 for all-trans retinoic acid, 12.7 for 9-cis retinoic acid, 29.6 for 13-cis retinoic acid, and 8.7 for DMSO. In comparison to the IC50 value for the P19 cell line, a similar IC50 value was obtained with 3T3 cells for ethanol, but there was a 2-fold increase for DMSO. The retinoids were not cytotoxic to 3T3 cells at the concentrations tested. This newly developed test is capable of detecting differentiation-inducing compounds at non-cytotoxic concentrations within 4 days. It offers a method for detecting chemicals with specific toxicological mechanisms, such as the retinoids, which could provide additional information in embryotoxicity testing as different promoters could be employed. Here, we report the use of this novel test system for the successful analysis of DMSO and three retinoids with different in vivo teratogenic potentials.

Background: Oxaliplatin and cisplatin are widely used in cancer chemotherapy, however, their clinical efficiency is often limited by the development of resistance., Materials and Methods: We examined different mechanisms of resistance in the human teratocarcinoma cell line 2102EP following exposure to cisplatin or oxaliplatin. Cells were exposed ten times with IC90-doses of 30 microM cisplatin and 50 microM oxaliplatin, respectively. Different cell clones were tested for expression of resistance using the SRB-assay. Moreover, resistance mechanisms in terms of drug uptake, platinum-adduct formation, GSH metabolism, DNA mismatch repair and p53 protein function were investigated., Results: Three cisplatin cell clones with significant resistance factors of 2.0 to 2.6 were found. Two oxaliplatin cell clones showed only weak resistance, with resistance factors of 1.6 and 1.7, respectively. In all three cisplatin-exposed cell clones a decreased cellular uptake of cisplatin was found. Furthermore, mechanisms of DNA damage tolerance may also play a role in the development of cisplatin-resistance in these cells. However, only two cell clones showed a decreased level of platinum-DNA-adducts. An increased DNA-repair of platinum-DNA adducts was not seen. In addition, no differences in expression of mismatch-repair proteins MSH2 and MLH1, tumor suppressor protein p53, or glutathione metabolism were found. However, significant resistance mechanisms for the observed oxaliplatin resistance could not be identified, although in one oxaliplatin-exposed cell clone, there was some evidence that a decreased cellular uptake of oxaliplatin may contribute to the observed low level resistance., Conclusions: The data add weight to the hypothesis that resistance mechanisms following oxaliplatin exposure may be similar to cisplatin. The precise mechanisms of resistance in the oxaliplatin-resistant cell clones are still not fully understood and current studies are underway to further eluciate this finding.

Nitrofen is a diphenyl ether herbicide that produces a spectrum of fetal abnormalities in rodents. To characterize the molecular mechanisms of nitrofen-mediated birth defects at the cellular level, we explored its effects on undifferentiated P19 teratocarcinoma cells. Nitrofen induces a time-dependent cell death of P19 cells that is associated with increases in TUNEL-positivity and caspase-3 cleavage suggesting that nitrofen induces P19 cell apoptosis. In addition, the increase in TUNEL-positive cells was inhibited with zVAD-fmk, suggesting that nitrofen induces a caspase-dependent apoptosis. Nitrofen treatment was associated with increased p38 MAP kinase activity, though pretreatment of cells with multiple p38 inhibitors did not affect nitrofen-mediated caspase-3 cleavage, suggesting caspase-3 cleavage is p38-independent. Nitrofen induced a dose-dependent increase in reactive oxygen species (ROS), which was accompanied by a decrease in the ratio of reduced/oxidized glutathione, indicating that nitrofen alters the cellular redox state of these cells. Furthermore, pretreatment of cells with N-acetyl cysteine gave a dose- and time-dependent reduction of caspase-3 cleavage, supporting the observations that caspase-3 cleavage is cell-redox-dependent. Therefore, nitrofen induces P19 cell apoptosis that is cell-redox-dependent and is associated with increases in p38 activity and ROS and may play a role in nitrofen-mediated birth defects.

Inhibition of the proteasomal pathway for degrading abnormal proteins leads to protein aggregation, increased oxidative damage and increased protein nitration. We now show that interference with polyubiquitination has similar consequences. Expression of a dominant-negative mutant form of ubiquitin (K48R) in NT-2 and SK-N-MC cells caused decreased cell growth rates and increased oxidative damage (protein carbonyls and lipid peroxidation), nitric oxide production and elevated protein nitration. It also rendered cells highly sensitive to 4-hydroxy-2,3-trans-nonenal, a neurotoxic end-product of lipid peroxidation, hydrogen peroxide and deprivation of growth factors. Overexpression of wild-type ubiquitin did not produce these effects. Our data show that interference with the ubiquitin-proteasome pathway at a different point and by a different mechanism can produce many of the common features of human neurodegenerative diseases, such as increased lipid peroxidation, protein oxidation and protein nitration. We suggest that defects in this pathway at multiple points could produce the common features of neurodegenerative diseases, and that more such defects remain to be discovered.

Alzheimer's disease (AD) brain reveals high rates of oxygen consumption and oxidative stress, altered antioxidant defences, increased oxidized polyunsaturated fatty acids, and elevated transition metal ions. Mitochondrial dysfunction in AD is perhaps relevant to these observations, as such may contribute to neurodegenerative cell death through the formation of reactive oxygen species (ROS) and the release of molecules that initiate programmed cell death pathways. In this study, we analyzed the effects of beta-amyloid peptide (Abeta) on human teratocarcinoma (NT2) cells expressing endogenous mitochondrial DNA (mtDNA), mtDNA from AD subjects (AD cybrids), and mtDNA from age-matched control subjects (control cybrids). In addition to finding reduced cytochrome oxidase activity, elevated ROS, and reduced ATP levels in the AD cybrids, when these cell lines were exposed to Abeta 1-40 we observed excessive mitochondrial membrane potential depolarization, increased cytoplasmic cytochrome c, and elevated caspase-3 activity. When exposed to Abeta, events associated with programmed cell death are activated in AD NT2 cybrids to a greater extent than they are in control cybrids or the native NT2 cell line, suggesting a role for mtDNA-derived mitochondrial dysfunction in AD degeneration.

We have previously shown that an F9 teratocarcinoma retinoic acid receptor beta(2) (RARbeta(2)) knockout cell line exhibits no growth arrest in response to all-trans-retinoic acid (RA), whereas F9 wild type (Wt), F9 RARalpha(-/-), and F9 RARgamma(-/-) cell lines do growth arrest in response to RA. To examine the role of RARbeta(2) in growth inhibition, we analyzed the cell cycle regulatory proteins affected by RA in F9 Wt and F9 RARbeta(2)(-/-) cells. Flow microfluorimetry analyses revealed that RA treatment of F9 Wt cells greatly increased the percentage of cells in the G1/G0 phase of the cell cycle. In contrast, RA did not alter the cell cycle distribution profile of RARbeta(2)(-/-) cells. In F9 Wt cells, cyclin D1, D3, and cyclin E protein levels decreased, while cyclin D2 and p27 levels increased after RA treatment. Compared to the F9 Wt cells, the F9 RARbeta(2)(-/-) cells exhibited lower levels of cyclins D1, D2, D3, and E in the absence of RA, but did not exhibit further changes in the levels of these cell cycle regulators after RA addition. Since RA significantly increased the level of p27 protein (approximately 24-fold) in F9 Wt as compared to the F9 RARbeta(2)(-/-) cells, we chose to study p27 in greater detail. The p27 mRNA level and the rate of p27 protein synthesis were increased in RA-treated F9 Wt cells, but not in F9 RARbeta(2)(-/-) cells. Moreover, RA increased the half-life of p27 protein in F9 Wt cells. Reduced expression of RARbeta(2) is associated with the process of carcinogenesis and RARbeta(2) can mediate the growth arrest induced by RA in a variety of cancer cells. Using both genetic and molecular approaches, we have identified some of the molecular mechanisms, such as the large elevation of p27, through which RARbeta(2) mediates these growth inhibitory effects of RA in F9 cells.

Clinically manifest hyperthyroidism is a rare paraneoplastic syndrome in patients with excessive HCG production due to testicular cancer. A 40-year-old patient with right testicular cancer (teratoma, embryonal cell carcinoma), diffuse pulmonary metastases and high serum HCG levels presented with symptomatic hyperthyroidism. The patient received immediately thyrostatic therapy and 4 cycles of PEI chemotherapy (Cisplatin, Etoposide, Ifosfamide). Thyroid function had returned to normal by the beginning of the second course of chemotherapy. After right orchiectomy and resection of residual pulmonary masses which revealed vital tumor cells, two additional courses of chemotherapy were performed. The patient is well and without evidence of disease 11 months after therapy. All patients with testicular cancer and excessive HCG production should be evaluated for biochemical and clinical signs of hyperthyroidism and treated accordingly with antithyroidal medication and immediate cytoreductive chemotherapy.

4-Hydroxy-2,3-trans-nonenal (HNE) is a neurotoxic unsaturated aldehyde end-product of lipid peroxidation. The addition of HNE to NT-2 and SK-N-MC cell lines induces apoptosis and we now investigated the time-course of events occurring prior to apoptosis. Treatment of both NT-2 and SK-N-MC cell lines with HNE led to HNE association with the proteasome, increased levels of protein carbonyls and ubiquitinated proteins, and decreased proteasomal function. There was also decreased metabolic activity, cytochrome c release and activation of caspase 3, followed by apoptotic changes including chromatin condensation, cell shrinkage and DNA fragmentation and laddering. Overexpression of mutant superoxide dismutase 1 proteins associated with amyotrophic lateral sclerosis decreased proteasomal activities in the absence of HNE and accelerated the apoptosis induced by HNE. By contrast, overexpression of wild-type superoxide dismutase 1 did not affect basal levels of proteasomal activity. The data suggest that accumulation of ubiquitinated proteins and impairment of proteasomal function are important events in HNE toxicity. We propose that the proteasomal system is a significant target of HNE neurotoxicity in a wide range of neurodegenerative diseases, especially if abnormal proteins are being expressed.

We prepared small unilamellar liposomes derivatised with single chain antibody fragments specific for the ED-B domain of B-fibronectin. This extracellular matrix associated protein is expressed around newly forming blood vessels in the vicinity of many types of tumours. The single chain antibody fragments were functionalised by introduction of C-terminal cysteines and linked to liposomes via maleimide groups located at the terminal ends of poly(ethylene glycol) modified phospholipids. The properties of these anti-ED-B single chain antibody fragments-liposomes were analysed in vitro on ED-B fibronectin expressing Caco-2 cells and in vivo by studying their biodistribution and their therapeutic potential in mice bearing subcutanous F9 teratocarcinoma tumours. Radioactively labelled ((114m)Indium) single chain antibody fragments-liposomes accumulated in the tumours at 2-3-fold higher concentrations during the first 2 h after i.v. injection compared to unmodified liposomes. After 6-24 h both liposome types were found in similar amounts (8-10% injected dose g(-1)) in the tumours. Animals treated i.v. with single chain antibody fragments-liposomes containing the new cytotoxic agent 2'-deoxy-5-fluorouridylyl-N(4)-octadecyl-1-beta-D-arabinofuranosylcytosine (30 mg kg(-1) per dose, five times every 24 h) showed a reduction of tumour growth by 62-90% determined on days 5 and 8, respectively, compared to animals receiving control liposomes. Histological analysis revealed a marked reduction of F9 tumour cells and excessive deposition of fibronectin in the extracellular matrix after treatment with single chain antibody fragments-2-dioxy-5-fluorouridylyl-N(4)-octadecyl-1-beta-D-arabinofuranosylcytosine-liposomes. Single chain antibody fragments-liposomes targeted to ED-B fibronectin positive tumours therefore represent a promising and versatile novel drug delivery system for the treatment of tumours., (Copyright 2002 Cancer Research UK)

Accumulating data suggest a central role for mitochondria and oxidative stress in neurodegenerative apoptosis. We previously demonstrated that amyloid-beta peptide 25-35 (Abeta 25-35) toxicity in cultured cells is mediated by its effects on functioning mitochondria. In this study, we further explored the hypothesis that Abeta 25-35 might induce apoptotic cell death by altering mitochondrial physiology. Mitochondria in Ntera2 (NT2 rho+) human teratocarcinoma cells exposed to either staurosporine (STS) or Abeta 25-35 were found to release cytochrome c, with subsequent activation of caspases 9 and 3. However, NT2 cells depleted of mitochondrial DNA (rho0 cells), which maintain a normal mitochondrial membrane potential (Deltapsi(m)) despite the absence of a functional electron transport chain (ETC), demonstrated cytochrome c release and caspase activation only with STS. We further observed increased reactive oxygen species (ROS) production and decreased reduced glutathione (GSH) levels in rho+ and rho0 cells treated with STS, but only in rho+ cells treated with Abeta 25-35. We conclude that under in vitro conditions, Abeta can induce oxidative stress and apoptosis only when a functional mitochondrial ETC is present.

Angiogenic processes depend on the precise coordination of different cell types and a complex exchange of signals, many of which derive from new specific components of the provisional, angiogenesis-related, extracellular matrix (ECM). Angiogenesis-associated ECM components thus represent appealing targets for the selective delivery of therapeutic molecules to newly forming tumor vessels. Results of a previous study indicated that a high affinity recombinant antibody (L19) to ED-B, a domain contained in the angiogenesis-associated isoform of fibronectin (B-FN), selectively and efficiently targets tumor vessels. The present study shows that a fusion protein between L19 and interleukin 2 (L19-IL-2) mediates the selective delivery and concentration of IL-2 to tumor vasculature, thereby leading to a dramatic enhancement of the therapeutic properties of the cytokine. By contrast, IL-2 fused to an irrelevant recombinant antibody used as a control fusion protein showed neither accumulation in tumors nor therapeutic efficacy. Tumors in mice treated with L19-IL-2 were significantly smaller compared to those in animals treated with saline, the control fusion protein, or IL-2 alone (P =.003,.003, and.002, respectively). Moreover, no significant differences in size were observed among the tumors from the different control groups (using the control fusion protein, a mixture of IL-2 and L19, or saline alone). Immunohistochemical analysis of tumor infiltrates demonstrated a significantly higher number of T lymphocytes, natural killer cells, and macrophages, as well as increased interferon-gamma (IFN-gamma) accumulation, in tumors from animals treated with L19-IL-2 compared to tumors from control groups. The fact that ED-B is 100% homologous in human and mouse, thus ensuring that L19 reacts equally well with human and murine antigen, should ultimately expedite transfer of this reagent to clinical trials.

p34(cdc2) is a protein kinase that plays an important role in the control of the cell cycle and regulation of activity of the tumor suppressor gene. Previously, we demonstrated that cisplatin (CDDP) induced growth suppression resulting in differentiation of teratocarcinoma F9 cells. In the present study, we investigated the mechanism of cell cycle retardation by CDDP in F9 cells, focusing on p34(cdc2). After the induction of differentiation with CDDP, F9 cells were arrested at the late S+G2/M. After treatment with CDDP, the level of the expression of cdc2 mRNA did not change. However, the half-life of p34(cdc2) was greatly reduced, thus, the level of p34(cdc2) protein was decreased. These findings suggest that the cell cycle arrest by CDDP is due partly to the induced instability of p34(cdc2) in F9 cells.

We present a case of large fungating scrotal mass in a 18-year-old male. Investigations revealed non-seminomatous germ cell tumor with retroperitoneal nodes. Systemic chemotherapy was followed by excision of the mass. This rare presentation is reported here., (Copyright 2002 S. Karger AG, Basel)

Teratocarcinomas are tumors that arise from primordial germ cells and are readily curable with DNA-damaging chemotherapeutic drugs. Teratocarcinoma cells ex vivo in tissue culture are also relatively chemosensitive and undergo apoptotic death in response to DNA damage. We have previously hypothesized that the observed sensitivity of this tumor type to DNA damage is related to high basal expression of wild-type p53 protein. We have now addressed this issue by characterizing the DNA damage response of isogenic teratocarcinoma cells that differ only in their level of expression of wild-type p53 protein. We find a clear p53 dose-response relationship in these cells for rapid apoptosis following DNA damage that correlates with diminished colony formation in clonogenic survival assays. These results suggest that strategies to increase basal wild-type p53 protein expression prior to treatment with DNA-damaging drugs may improve curability in other tumor types.

Organotin compounds, particularly tri-organotin, have demonstrated cytotoxic properties against a number of tumor cell lines. On this basis, triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29), a quinolizidine derivative, was synthesized and developed as a potential antitumor agent. This tin-derived compound exhibited potent antiproliferative effects on three different human cancer cell lines: teratocarcinoma of the ovary (PA-1), colon carcinoma (HCT-8) and glioblastoma (A-172). Cytotoxic activity was assessed by MTT and cell count assays during time course experiments with cell recovery after compound withdrawal. Significant cell growth inhibition (up to 95% in HCT-8 after 72 h of exposure), which also persisted after drug-free medium change, was reported in all the cell lines by both assays. In addition, the cytocidal effects exerted by IST-FS 29 appeared more consistent with necrosis or delayed cell death, rather than apoptosis, as shown by morphologic observations under light microscope, DNA fragmentation analysis and flow cytometry. In the attempt to elucidate whether this compound might affect genes playing a role in G1/S phase transition, the expressions of p53, p21(WAF1), cyclin D1 and Rb, mainly involved in response to DNA-damaging stress, were analyzed by Western blot. Heterogeneous patterns of expression during exposure to IST-FS 29 were evidenced in the different cell lines suggesting that these cell-cycle-related genes are not likely the primary targets of this compound. Thus, the present data seem more indicative of a direct effect of IST-FS-29 on macromolecular synthesis and cellular homeostasis, as previously hypothesized for other organotin complexes.

Taurine acts as an antioxidant able to protect neurons from free radical-mediated cellular damage. Moreover, it modulates the immune response of astrocytes that participate in neurodegenerative processes. The objective of this study was to examine whether taurine can prevent or attenuate the host inflammatory response induced by the xenotransplantation of neurons derived from the human teratocarcinoma cell line (hNT neurons). Male Sprague-Dawley rats were treated IP with either saline or taurine. Animals from both groups were perfused on the 4th or 11th day and the saline or taurine was administered from the start of the study until the day prior to sacrifice. The brains were processed immunohistochemically using antibodies against glial fibrillary acidic protein (GFAP), microglia (OX42), and human nuclear matrix antigen (NuMA). In the saline group, NuMA labeling revealed small grafts on the 4th day and no surviving cells on the 11th day. However, in the group that received taurine there were surviving grafts at both time points. Strong immunoreactivity for GFAP and OX42 was detected in the saline group surrounding the transplant. These effects were reduced in animals receiving taurine. Taken together, these results demonstrated that taurine was able to facilitate graft survival and attenuate the immune response generated by the xenograft.

We report a very rare case of teratocarcinosarcoma of the nasal cavity showing a favorable prognosis. The patient was a 66-year-old man with a mass completely obstructing the right nasal cavity. Subsequently, extirpation of the mass and Denker-Watsuji operation were performed, and the patient was treated with a combination of radiation therapy and chemotherapy. Neither recurrence nor distant metastasis was observed during follow-up lasting 30 months. Histologic examination of the resected mass revealed several tissue elements including columnar and squamous epithelia with atypia, smooth muscle cells with rare mitotic activity, and neuroectodermal tissue. The glandular epithelium and smooth muscle cells were reminiscent of a primitive intestinal organoid structure, suggestive of teratomatous tumorigenesis. Our case and a review of the literature indicate that the absence of invasiveness to the stroma or surrounding tissue is closely related to a favorable prognosis.

Retinoic acid (RA) plays a pivotal role during vertebrate development, both as morphogen and as potent teratogen. While RA function in axial development has been extensively studied, little is known about the genetic control of RA teratogenicity. The knockout of the homeobox gene goosecoid in the mouse revealed similarities to RA induced embryopathy. We show that RA treatment of mouse gastrula embryos in vitro and of E10.5 embryos in utero led to a rapid but transient down-regulation of goosecoid expression. Repression was dependent on retinoid X receptors (RXR). BMP-4 was repressed by RA-treatment as well, both in embryos and in F9 teratocarcinoma cells. Our data suggest that both goosecoid and BMP-4 function as mediators of RA teratogenicity in mouse embryos.

We reported two cases of chemotherapy-refractory testicular cancer treated with all trans-retinoic acid (ATRA). Case 1. A 21-year-old male patient underwent salvage surgery for lung metastasis which had developed after treatment with three different cisplatin-based chemotherapy regimens for malignant teratoma. After recovery from surgery, he was treated with oral ATRA at daily dose 80 mg/m2 for four weeks. Case 2. A-45-year-old patient suffered from lung metastasis after orchiectomy for teratocarcinoma. The patient failed to achieve a complete response despite two different cisplatin-based chemotherapy and high dose chemotherapy regimens with bone marrow rescue. He was treated with oral ATRA for five weeks. Both patients showed disease progression with increase in tumor size and elevation of tumor marker during ATRA therapy. Side effects were acceptable except the headache in Case 2, who needed a dose reduction of ATRA. In conclusion, oral ATRA with this dose failed to show clinical antitumor activity in patients with refractory testicular cancer.

The antiepileptic drug valproic acid (VPA) is an established human teratogen that affects neural development in human fetuses exposed to the drug during early pregnancy. The development of simple cell culture models that reflect normal neuronal development will be useful for the screening of neural developmental toxicity of new drugs and for the identification of the specific molecular targets for the teratogenic action. The present study tests a human NT2 cell line for its suitability to serve as such a model. Treatment with VPA, but not with its non-teratogenic-analogue, 2-isopropylpentanoic acid (IPPA), inhibited cell growth, had a cytotoxic effect and blocked retinoic acid-induced neuronal differentiation of NT2 cells. Differentiation was evaluated by immunostaining for neurofilament proteins, microtubule associated proteins-2 (MAP-2), tau and neural cell adhesion molecule, NCAM. However once differentiation had taken place, VPA did not revert this process, although it did affect neuronal aggregation and neurite fasciculation. These results suggest that the human NT2 cell line might be useful for identifying drugs affecting neuronal development in humans.

Testicular cancers respond favorably to chemotherapy with the platinum-containing drug cis-diamminedichloroplatinum(II) (cisplatin). One factor that could explain the efficacy of cisplatin is the low frequency of p53 mutations observed in this tumor type. The present study examines the p53-mediated responses in murine testicular teratocarcinoma cells exposed to the drug. Cisplatin treatment of teratocarcinoma cells with a wild-type p53 gene resulted in accumulation of the p53 protein through posttranscriptional mechanisms; induction of p53-target genes was also observed. Drug treatment resulted in rapid apoptosis in p53-wild-type cells but not in p53(-/-) teratocarcinoma cells. In the latter cells, cisplatin exposure caused prolonged cell cycle arrest accompanied by induction of the p21 gene. Clonogenic assays demonstrated that the p53 mutation did not confer resistance to cisplatin. These experiments suggest that cisplatin inhibits cellular proliferation of testicular teratocarcinoma cells by two possible mechanisms, p53-dependent apoptosis and p53-independent cell cycle arrest.

Poor solubility of paclitaxel constitutes an important limitation to its administration to cancer patients. In order to increase solubility, while preserving cytotoxicity, a new paclitaxel derivative substituted at the 2' position of the side chain was prepared. The first step consisted in the introduction of a glutaryl group at the 2' position by esterification of the free hydroxyl followed by an amidation with 6-amino hexanol previously attached to a glucuronide group. The new water soluble derivative was cytotoxic in vitro against an ovarian teratocarcinoma cell line.

We report one male with testicular cancer and one with lymphoma who received transplants more than 20 years ago. Both are well.

We established a xenograft line of human teratocarcinoma (TC-1) and characterized the pluripotency of differentiation of the neoplastic cells. A teratocarcinoma specimen obtained from a primary mediastinal lesion (22-year-old male patient) was inoculated subcutaneously into severe combined immunodeficient (SCID) mice. The carcinoma formed tumors in the mice. We established a xenograft line by serial passage of the tumor in vivo. The primary tumor was composed of papillary and pseudoglandular nests of highly atypical epithelial cells with foci of glomeruloid structures. The metastatic cells showed apparent production of mucin and differentiation to striated muscle. The xenograft line TC-1 retained the basic histopathological features seen in the primary and metastatic cells. The xenograft line showed focal differentiation to cartilage through serial passages. Immunohistochemical studies with anti-alpha-fetoprotein (AFP) demonstrated positive immunoreactivity on the TC-1 cells. Serum AFP levels were also elevated in the TC-1-bearing SCID mice. The human teratocarcinoma xenograft line TC-1 will be useful for studying the differentiation mechanism in human totipotent stem cells.

Objective: The antitumor effect of CPT-11, a camptothecin derivative, on two human testicular embryonal carcinomas (TTSC-2 and TTSC-3) heterotransplanted into-nude mice was studied., Materials and Methods: Tumor-bearing nude mice were given daily intraperitoneal injections of the anticancer drugs in 0.1 ml saline 3 times at 3-day intervals. At the end of the experiments tumors were resected and subjected to light-microscopic observation., Results: When 10, 30 and 50 mg/kg of CPT-11 was administered to tumor-bearing mice intraperitoneally, the antitumor effect of CPT-11 was observed dose-dependently in both TTSC-2 and TTSC-3. When 30 mg/kg of CPT-11 was administered in combination with CDDP, complete tumor regression was observed in both TTSC-2 and TTSC-3 tumors. Histological findings correlated well with the decrease in tumor volume of treated tumors. No mice died after treatment with CPT-11 in a single-agent and combination chemotherapy., Conclusion: Chemotherapy with CPT-11 was an effective and safe method against human testicular tumors heterotransplanted in nude mice.

Background: Sparse data are available with regard to the incidence, clinical characteristics, therapeutic management and prognosis of male patients with germ cell tumors, who relapse more than two years after completion of cisplatin-based chemotherapy., Patients and Methods: A review of 530 patients treated at two institutions from 1978 to April 1994 was conducted. Twenty-five cases of late relapse were identified. Cumulative risk of late relapse was calculated according to the Kaplan-Meier method., Results: 418 of 523 patients (80%) who received their first-line treatment at our institutions were relapse-free at two years. Among these 418 patients 18 cases (4.3%) developed a late relapse. The cumulative risk of late relapse was 1.1% at five years and 4.0% at ten years excluding patients with prior early relapses who carried risks of 9.4% and 29%, respectively (P < 0.0001). No case of late relapse was observed among patients receiving adjuvant chemotherapy. The risk of late relapse was lower in patients with good-risk non-seminomatous germ cell tumors than in poor-risk patients according to Medical Research Council criteria (P < 0.01). Seven further patients were referred from other institutions for treatment of late relapse. At a median follow-up of 38 months (range, 3 to 121) after treatment of late relapse 9 of 25 patients (36%) are continuously disease-free. Six of these nine patients had surgical resection of carcinoma or teratoma as a component of their therapy., Conclusion: The incidence of late relapse after cisplatin-based chemotherapy of germ cell tumors is related to initial tumor burden and is somewhat higher than previously expected. Chemotherapy seems to have only minor curative potential, but localized resectable disease can be cured by surgery. Annual follow-up evaluations allow to detect the majority of late relapses at an asymptomatic stage and should be extended throughout the patient's life.

Bone cells involved in the replacement of cartilage by bone in the endochondral ossification process are known to enter via the medullar pathway. A hypothesis for the development of osteoblasts from chondroblasts was investigated by analyzing the phenotypic characteristics of the 3/A/1D-1M cell line derived from endochondral bone ossicle which was formed after in vivo transplantation of 3/A/1D-1 chondroprogenitor mouse teratocarcinoma cells. The 3/A/1D-1M cell cultures exhibited a triphasic evolution: after reaching confluence (day 3), cultures developed well-delimited cell clusters (days 6-8), which ultimately were organized into multilayered nodules (days 12-15). Electron-microscopic examination of such nodules at day 18 showed the presence of needle-shaped crystals associated with collagen fibrils in the extracellular space. The kinetics of collagen expression, investigated by an immunofluorescence staining procedure showed that, while confluent cultures mainly expressed type III collagen (70% of cells) with some type I (30-40% of cells) and V (30-40% of cells), the type I collagen became the major isoform beginning with day 6. From day 6 onwards, NP40-extracted alkaline phosphatase (AP) activity appeared concomitantly to cell cluster formation, and reached 160 nmol/min/mg of protein at the stage of nodule maturation (day 15). The strong inhibition of enzymatic activity by levamisole and L-homoarginine (IC50 = 0.9 microM and 5 mM, respectively) and its rapid heat inactivation at 56 degrees C (IT50 = 90 s), revealed the bone specificity of AP expressed by 3/A/1D-1M cells. In confluent cultures, brief exposure to parathyroid hormone (10 nM), known to be a bone-resorbing agent, showed a 60% increase in the intracellular cAMP level. In addition, while producing mRNA for the bone-specific protein osteocalcin, 3/A/1D-1M cells also produced type II procollagen mRNA, known to be the major cartilage-related characteristic. This in vitro study demonstrates that the 3/A/1D-1M clonal cell line, originating from 3/A/1D-1 chondroprogenitor cells after in vivo passage, was able to develop differentiated osteoblastic properties as well as the residual expression of the major chondrocytic RNA messenger.