Ultrasound-Guided Intramural Inoculation of Orthotopic Bladder Cancer Xenografts: A Novel High-Precision Approach

Orthotopic bladder cancer xenografts are essential for testing novel therapies and molecular manipulations of cell lines in vivo. Current xenografts rely on tumor cell inoculation by intravesical instillation or direct injection into the bladder wall. Instillation is limited by the lack of cell lines that are tumorigenic when delivered in this manner. The invasive model inflicts morbidity on the mice by the need for laparotomy and mobilization of the bladder. Furthermore this procedure is complex and time-consuming. Three bladder cancer cell lines (UM-UC1, UM-UC3, UM-UC13) were inoculated into 50 athymic nude mice by percutaneous injection under ultrasound guidance. PBS was first injected between the muscle wall and the mucosa to separate these layers, and tumor cells were subsequently injected into this space. Bioluminescence and ultrasound were used to monitor tumor growth. Contrast-enhanced ultrasound was used to study changes in tumor perfusion after systemic gemcitabine/cisplatin treatment. To demonstrate proof of principle that therapeutic agents can be injected into established xenografts under ultrasound guidance, oncolytic virus (VSV) was injected into UM-UC3 tumors. Xenograft tissue was harvested for immunohistochemistry after 23–37 days. Percutaneous injection of tumor cells into the bladder wall was performed efficiently (mean time: 5.7 min) and without complications in all 50 animals. Ultrasound and bioluminescence confirmed presence of tumor in the anterior bladder wall in all animals 3 days later. The average tumor volumes increased steadily over the study period. UM-UC13 tumors showed a marked decrease in volume and perfusion after chemotherapy. Immunohistochemical staining for VSV-G demonstrated virus uptake in all UM-UC3 tumors after intratumoral injection. We have developed a novel method for creating orthotopic bladder cancer xenograft in a minimally invasive fashion. In our hands this has replaced the traditional model requiring laparotomy, because this model is more time efficient, more precise and associated with less morbidity for the mice.     

Augmentative effect of Nocardia rubra cell-wall skeleton (N-CWS) on lymphokine-activated killer (LAK) cell induction

Summary The present study elucidated that N-CWS augments the cytolytic activity against 3LL tumor cells of LAK cells from N-CWS-immunized mice administered i.p. with rIL-2. This augmentative effect of N-CWS was not seen when the LAK cells were prepared from normal mice. The cytolytic activity was predominantly expressed in the NAPC prepared from the site of injection of rIL-2, and repeated administrations of rIL-2 were required to induce and maintain this potent cytolytic activity in vivo. Serological analysis revealed that the LAK cells were positive for Thy 1.2 and asialo GM1 antigens and that they were not classical CTL or NK cells. The administration of rIL-2 statistically prolonged the MST of mice bearing LAK-sensitive 3LL cells but not the MST of mice bearing LAK-resistant EL-4 leukemia. Furthermore, combination therapy with N-CWS and rIL-2 prolonged the MST of the mice more than the therapy with rIL-2 alone. These results suggest that LAK cells potentiated with N-CWS would be useful for immunotherapy of malignant neoplasms. Abbreviations used: N-CWS, Nocardia rubra cell-wall skeleton; rIL-2, recombinant interleukin 2; LAK, lymphokine-activated killer; RPMI 1640, Roswell Park Memorial Institute 1640; FCS, fetal calf serum; TCM, tumor culture medium; PC, peritoneal cells; NAPC, nonadherent PC; APC adherent PC; MST, mean survival time; NK, natural killer; E:T ratios, effector to target ratios; Poly I:C, polyinosinic-polycytidylic acid; CTL, cytotoxic T lymphocytes; RLNC, regional lymphnode cells     

Hierarchy of in vitro sensitivity and resistance of tumor cells to cytotoxic effector cells,cytokines, drugs and toxins

Summary Drug resistance of tumor cells has led to the development of other therapeutic modalities including biological response modifiers, lymphokine-activated killer cells (LAK), and cytokines alone and in combination. The premise of these alternative modalities is that drug resistance can be overcome by other cytotoxic agents or cytotoxic effector cells. However, the relationship between tumor cell sensitivity to these different agents and the cytotoxicity caused by drugs is not known or well understood. Thus, understanding the relationship between these different systems of tumor cell cytotoxicity is essential for optimal therapeutic intervention. To this end, we compared the tumor cell cytotoxicity mediated by recombinant tumor necrosis factor (rTNF), cytotoxic effector cells (natural killer cells, monocytes, LAK cells), chemotherapeutic drugs, and microbial toxins. Human tumor cell lines sensitive and resistant to rTNF or drugs were used to evaluate the effectiveness of the other cytotoxic modalities. Sensitivity was considered as tumor cell cytotoxicity above 15% while resistance refers to that below 10%. Cell lines tested consisted of several histological types such as brain, lung, colon and ovarian tumors. In our experiments, cell lines made resistant to rTNF by coculture were also relatively resistant to unactivated monocytes and their supernatants. These lines were sensitive to all other methods tested including activated monocytes, natural killer and LAK cells, drugs, and toxins. The tumor lines naturally resistant to rTNF were found to have various degrees of sensitivity and resistance to these other systems. Upon the analysis of our data, a pattern emerged that suggested a hierarchy of sensitivity and resistance of the tumor cells to the cytotoxic mechanisms explored. From a majority of cell lines resistant to rTNF to a minority of lines resistant to LAK, we found an interesting gradation of sensitivity and/or resistance to the other cytotoxic modalities employed. The hypothesis of an underlying common mechanism of action within these systems is discussed.Supported in part by grant CA43 121 from the Department of Health and Human services, NIH, and NRSA clinical and fundamental immunology training grant A107 126, NIH (J. S.), and in part by a grant from the Concern Foundation, Los Angeles and a gift from the Boiron Foundation     

Specificity of adoptive chemoimmunotherapy of established syngeneic tumors

To examine the specificity of adoptive chemoimmunotherapy (ACIT) of established syngeneic tumors, two noncross-reactive C57BL/6 tumors were studied: a Friend virus-induced tumor (FBL-3) and a chemically induced virus-negative tumor EL-4(G-). In vitro studies confirmed that these tumors are antigenically distinct by demonstrating that the cytotoxic responses of spleen cells from mice immunized in vivo and reexposed to tumor in vitro are immunologically specific. Studies of ACIT with cells from mice immunized in vivo demonstrated similar specificity. Mice receiving 5 x 10(6) FBL-3 on day 0 all died by day 13. Treatment on day 5 with cyclophosphamide (CY), 180 mg/kg, prolonged the median survival time (MST) to day 23. Treatment on day 5 with CY plus 2 x 10(7) normal nonimmune C57BL/6 cells or CY plus cells sensitized to EL-4(G-) had no additional effect on survival whereas 2 x 10(7) C57BL/6 cells sensitized to FBL-3 in vivo prolonged MST to day 64 and cured 13 of 32 mice. Similarly, mice given 2 x 10(5) EL-4(G-) on day 0 all died by day 16, and CY on day 5 prolonged the MST to day 22. As an adjunct to CY, 2 X 10(7) normal cells or cells sensitized to FBL-3 had a modest effect, prolonging the MST to days 37 and 36, respectively. However, treatment with CY plus 2 x 10(7) cells immune to EL-4(G-) cured 22 of 32 mice. The results demonstrate the immunologic specificity of ACIT of syngeneic tumors treated with immune syngeneic cells.     

Lymphokine-activated killer (LAK) and monocyte-mediated cytotoxicity on tumor cell lines resistant to antitumor agents

In this study we have examined the susceptibility of tumor cell lines exhibiting different patterns of resistance to chemotherapeutic agents, to the cytotoxic action of lymphokine-activated killer (LAK) cells and activated monocytes. The susceptibility of tumor cells with pleiotropic drug resistance to these cytotoxic mechanisms was not different from that of their parental, chemo-sensitive cell lines. Tumor lines used in this study included three human cell lines (LOVO N and LOVO/Dx, I-407 and I-407/Dx, MCF7 and MCF7a) selected for being resistant to doxorubicin and showing a pleiotropic pattern of resistance, and the murine ovarian reticulum cell sarcoma M5076 and its variants resistant to individual antitumor agents (cisplatin, cyclophosphamide and 5-aza-2'-deoxycytidine). These results demonstrate that drug-resistant tumor cell lines, irrespective of the pattern of resistance, were susceptible to the in vitro cytotoxicity mediated by LAK cells and activated monocytes with levels of lysis similar to those of parental chemosensitive lines. Moreover, freshly isolated tumor cells from ovarian cancer patients unresponsive to different chemotherapeutic treatments (operationally drug-resistant) were significantly killed in vitro by LAK cells. These findings support the concept that activated effector cells have the potential to complement conventional chemotherapy by eliminating drug-resistant tumor variants.     

Collateral Chemoresistance to Anti-Microtubule Agents in a Lung Cancer Cell Line with Acquired Resistance to Erlotinib

Various alterations underlying acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been described. Although treatment strategies specific for these mechanisms are under development, cytotoxic agents are currently employed to treat many patients following failure of EGFR-TKIs. However, the effect of TKI resistance on sensitivity to these cytotoxic agents is mostly unclear. This study investigated the sensitivity of erlotinib-resistant tumor cells to five cytotoxic agents using an in vitro EGFR-TKI-resistant model. Four erlotinib-sensitive lung adenocarcinoma cell lines and their resistant derivatives were tested. Of the resistant cell lines, all but one showed a similar sensitivity to the tested drugs as their parental cells. HCC4006ER cells with epithelial mesenchymal transition features acquired resistance to the three microtubule-targeting agents, docetaxel, paclitaxel and vinorelbine, but not to cisplatin and gemcitabine. Gene expression array and immunoblotting demonstrated that ATP-binding cassette subfamily B, member 1 (ABCB1) was up-regulated in HCC4006ER cells. ABCB1 knockdown by siRNA partially restored sensitivity to the anti-microtubule agents but not to erlotinib. Moreover, the histone deacetylase inhibitor entinostat sensitized HCC4006ER cells to anti-microtubule agents through ABCB1 suppression. Our study indicates that sensitivity of tumor cells to cytotoxic agents in general does not change before and after failure of EGFR-TKIs. However, we describe that two different molecular alterations confer acquired resistance to EGFR-TKIs and cytotoxic agents, respectively. This phenomenon should be kept in mind in selection of subsequent therapy after failure of EGFR-TKIs.     

A potent replicative delta-24 adenoviral vector driven by the promoter of human papillomavirus 16 that is highly selective for associated neoplasms

BACKGROUND: Several human epithelial neoplasms are associated with high-risk strains of human papillomavirus (HPV) such as cervical, anorectal, and other carcinomas. For some tumor types the current therapeutic tools are only palliative. Conditionally replicative adenoviruses (CRAds) are promising antineoplastic agents, which also can trigger confined antitumor effects. METHODS: We constructed a series of CRAds driven by the upstream regulatory promoter region (URR) of an Asian-American variant of HPV-16, which contained different mutations at the E1A region (dl1015 and/or Delta24) and wild-type. All vectors were tested in vitro for viral replication and cytotoxicity. Viral DNA replication and E1A expression were also assessed by quantitative PCR. Finally, we confirmed the antitumoral efficacy of this vector in injected and non-injected xenotransplanted cervical tumors in a murine model for tumor regression and survival studies. RESULTS: A vector denominated Ad-URR/E1ADelta24 displayed a potent cytopathic effect associated with high selectivity for HPV+ cell lines. We found that the oncolytic effect of this CRAd was comparable to Ad-wt or Ad-Delta24, but this efficacy was significantly attenuated in HPV- cell lines, an effect that was contributed by the URR promoter. Ad-URR/E1ADelta24 was very effective to control tumor growth, in both, injected and non-injected tumors generated with two different HPV+ cell lines. CONCLUSIONS: CRAd Ad-URR/E1ADelta24 is a highly selective vector for HPV+ cell lines and tumors that preserves the oncolytic efficacy of Ad-wt and Ad-Delta24. Our preclinical data suggest that this vector may be useful and safe for the treatment of tumors induced by HPV, like cervical cancers.     

CD49fhigh Cells Retain Sphere-Forming and Tumor-Initiating Activities in Human Gastric Tumors

Identification of gastric tumor-initiating cells (TICs) is essential to explore new therapies for gastric cancer patients. There are reports that gastric TICs can be identified using the cell surface marker CD44 and that they form floating spheres in culture, but we could not obtain consistent results with our patient-derived tumor xenograft (PDTX) cells. We thus searched for another marker for gastric TICs, and found that CD49f^high cells from newly-dissected gastric cancers formed tumors with histological features of parental ones while CD49f^low cells did not when subcutaneously injected into immunodeficient mice. These results indicate that CD49f, a subunit of laminin receptors, is a promising marker for human gastric TICs. We established a primary culture system for PDTX cells where only CD49f^high cells could grow on extracellular matrix (ECM) to form ECM-attaching spheres. When injected into immunodeficient mice, these CD49f^high sphere cells formed tumors with histological features of parental ones, indicating that only TICs could grow in the culture system. Using this system, we found that some sphere-forming TICs were more resistant than gastric tumor cell lines to chemotherapeutic agents, including doxorubicin, 5-fluorouracil and doxifluridine. There was a patient-dependent difference in the tumorigenicity of sphere-forming TICs and their response to anti-tumor drugs. These results suggest that ECM plays an essential role for the growth of TICs, and that this culture system will be useful to find new drugs targeting gastric TICs.     

Characterization of tumor reactivity of human V gamma 9V delta 2 gamma delta T cells in vitro and in SCID mice in vivo

Human Vgamma9Vdelta2 gammadelta T cells are selectively activated by bacterial phosphoantigens and aminobisphosphonates and exert potent cytotoxicity toward various tumor cells. In this study we have characterized the cytotoxic reactivity of gammadelta T cell lines established from healthy donors by stimulation with aminobisphosphonate alendronate toward melanoma MeWo and pancreatic adenocarcinomas Colo357 and PancTu1 lines in vitro and in vivo upon adoptive transfer into SCID mice. Lysis of all tumor cells was enhanced when gammadelta effector cells were preactivated with phosphoantigens. Recognition of MeWo was TCR dependent, as shown by anti-TCR Ab blockade, whereas only the phosphoantigen-mediated increased, but not the basal, lysis of Colo357 and PancTu1 was inhibited by anti-TCR Ab. Furthermore, lysis of Colo357, but not that of MeWo or PancTu1, was completely inhibited by the pan-caspase inhibitor zVAD, indicating different recognition and effector mechanisms involved in the gammadelta T cell/tumor cell interactions. Upon transfer into SCID mice, alendronate-activated gammadelta T cells given together with IL-2 and alendronate significantly prolonged the survival of SCID mice inoculated with human tumor cells. The best results were thus obtained when gammadelta T cells were repetitively given five times over a period of 30 days. With this protocol, human gammadelta T cells prolonged the mean survival of mice inoculated with MeWo melanoma from 28.5 to 87.3 days (p < 0.0001) and in the case of PancTu1 adenocarcinoma from 23.0 to 48.4 days (p < 0.0001). We conclude that an effective gammadelta T cell-based immunotherapy might require activation of endogenous gammadelta T cells with aminobisphosphonate (or phosphoantigen) and IL-2, followed by adoptive transfer of in vitro expanded gammadelta T cells.     

Breakthrough pulmonary Aspergillus fumigatus infection with multiple triazole resistance in a Spanish patient with chronic myeloid leukemia

BackgroundAn allogeneic hematopoietic cell transplantation (allo-HCT) patient presented with chronic pulmonary aspergillosis associated to pulmonary graft versus host disease (GVHD) and was treated for a long time with several antifungal agents that were administered as prophylaxis, combination therapies, and maintenance treatment. The patient suffered from a breakthrough invasive pulmonary aspergillosis due to Aspergillus fumigatus after long-term antifungal therapy.Material and methodsSeveral isolates were analyzed. First isolates were susceptible in vitro to all azole agents. However, after prolonged treatment with itraconazole and voriconazole a multiple azole resistant A. fumigatus isolate was cultured from bronchoalveolar lavage (BAL) when the patient was suffering from an invasive infection, and cavitary lesions were observed.ResultsAnalysis of the resistant mechanisms operating in the last strain led us to report the first isolation in Spain of an azole resistant A. fumigatus strain harboring the L98H mutation in combination with the tandem repeat (TR) alteration in CYP51A gene (TR-L98H). Long-term azole therapy may increase the risk of resistance selecting strains exhibiting reduced susceptibility to these compounds. However, since the isolates were genetically different the suggestion that could be made is that the resistance was not induced during the prolonged azole therapy but the patient might simply have acquired this resistant isolate from the environment, selected by the therapy.ConclusionsThese findings suggest that in all long-term treatments with antifungal agents, especially with azoles, repeated sampling and regular susceptibility testing of strains isolated is necessary as resistant isolates could be selected.     

In vivo natural antitumor resistance against murine EL-4 lymphoma cells in lethally irradiated syngeneic C57Bl/6 mice

Natural resistance has been detected in lethally irradiated C57Bl/6 (B6) mice inoculated intravenously with the ascites form of a syngeneic B6 leukemia. EL-4 cells were injected into lethally irradiated (800 R) B6 mice and tumor cell proliferation was evaluated by 125IUdR uptake in different organs 4 days after the challenge. Differential growth of lymphoma cells was observed when young mice were injected as compared with older mice and when mice were treated with agents known to interfere with natural resistance (e.g., poly(I:C), FLV-P, carrageenan, cyclophosphamide, high doses of irradiated cells). Similar results were obtained by measuring rapid clearance of 125IUdR-labeled EL-4 cells from lungs of intact B6 mice. In vivo cold competition studies, employing EL-4 and several other tumor lines of the same or different haplotype, showed that only EL-4 and RBL-5 cells were capable of inhibiting syngeneic resistance against EL-4 tumor. On the contrary, YAC-1 lymphoma cells, the most susceptible target to natural killer-mediated cytotoxicity in vitro, did not compete. These results suggest that EL-4 cells express membrane determinants not detectable on normal H-2b parental bone marrow cells and are susceptible to natural resistance against hemopoietic tumor cells in lethally irradiated syngeneic B6 mice.     

Antitumor agents. Part 212. Bucidarasins A-C, three new cytotoxic clerodane diterpenes from Bucida buceras

As part of a study on antitumor agents from rainforest plants, four new clerodane diterpenes, bucidarasins A--D (1-4), were isolated from Bucida buceras. Their structures were elucidated from detailed 2D NMR analyses. Compounds 1-3 showed potent cytotoxicity against human tumor cell lines with IC(50) values of 0.5-1.9 microM. The potency was retained in drug resistant lines.     

Characterization of T cell lines and clones from SJL/J and (BALB/c x SJL/J)F1 mice specific for myelin basic protein

Lines of thymus-derived lymphocytes reactive against bovine myelin basic protein (BP) were established in vitro from SJL/J mice. These lines are stable in long-term culture and mediate inflammatory central nervous system (CNS) lesions and a low incidence of clinical experimental allergic encephalomyelitis (EAE) when injected into recipient SJL/J mice. The line cells proliferate in response to BP of bovine, rat, or mouse origin. Clones were derived from these lines, and the characteristics of these clones were analyzed. The clones express Thy-1, Ly-1, and L3T4 antigens and are negative for Ly-T2. The clones all proliferate in response to bovine BP, with different clones showing varying degrees of cross-reactivity between bovine, rat, and mouse BP. The proliferative response is MHC-restricted; antigen-presenting cells from I-As strains are required. Compatible with their phenotype as helper cells, some of the clones will provide help to primed B cells stimulating antibody production in an in vitro assay. When injected into recipients pretreated with pertussis and irradiation, clones that showed proliferation to mouse BP induced the development of inflammatory lesions in the CNS, with mortality of 28% of the recipients. T cell lines were also established in (BALB/c x SJL/J)F1 mice. In contrast to the homozygous SJL/J lines, these lines were highly encephalitogenic, inducing a high incidence of clinical and histologic EAE when injected in vivo.     

LncRNA MST1P2/miR‐133b axis affects the chemoresistance of bladder cancer to cisplatin‐based therapy via Sirt1/p53 signaling

Although bladder cancer is commonly chemosensitive to standard first‐line therapy, the acquisition of the resistance to cisplatin (DDP)‐based therapeutic regimens remains a huge challenge. Noncoding RNAs (ncRNAs), including long noncoding RNAs (lncRNAs) and microRNAs, have been reported to play a critical role in cancer resistance to DDP. Here, we attempted to provide a novel mechanism by which the resistance of bladder cancer to DDP treatment could be modulated from the perspective of ncRNA regulation. We demonstrated that lncRNA MST1P2 (lnc‐MST1P2) expression was dramatically upregulated, whereas miR‐133b expression was downregulated in DDP‐resistant bladder cancer cell lines, SW 780/DDP and RT4/DDP. Lnc‐MST1P2 and miR‐133b negatively regulated each other via targeting miR‐133b. Both lnc‐MST1P2 silence and miR‐133b overexpression could resensitize DDP‐resistant bladder cancer cells to DDP treatment. More important, miR‐133b could directly target the Sirt1 3′‐untranslated region to inhibit its expression. Inc‐MST1P2/miR‐133b axis affected the resistance of bladder cancer cells to DDP via Sirt1/p53 signaling. In conclusion, MST1P2 serves as a competing endogenous RNA for miR‐133b to counteract miR‐133b‐induced suppression on Sirt1, therefore enhancing the resistance of bladder cancer cells to DDP. MST1P2/miR‐133b axis affects the resistance of bladder cancer cells to DDP via downstream Sirt1/p53 signaling.     

The role of the CD134-CD134 ligand costimulatory pathway in alloimmune responses in vivo

The CD134-CD134 ligand (CD134L) costimulatory pathway has been shown to be critical for both T and B cell activation; however, its role in regulating the alloimmune response remains unexplored. Furthermore, its interactions with other costimulatory pathways and immunosuppressive agents are unclear. We investigated the effect of CD134-CD134L pathway blockade on allograft rejection in fully MHC-mismatched rat cardiac and skin transplantation models. CD134L blockade alone did not prolong graft survival compared with that of untreated recipients, and in combination with donor-specific transfusion, cyclosporine, or rapamycin, was less effective than B7 blockade in prolonging allograft survival. However, in combination with B7 blockade, long-term allograft survival was achieved in all recipients (>200 days). Moreover, this was synergistic in reducing the frequency of IFN-gamma-producing alloreactive lymphocytes and inhibiting the generation of activated/effector lymphocytes. Most impressively, this combination prevented rejection in a presensitized model using adoptive transfer of primed lymphocytes into athymic heart transplant recipients. In comparison to untreated recipients (mean survival time (MST): 5.3 +/- 0.5 days), anti-CD134L mAb alone modestly prolonged allograft survival (MST: 14 +/- 2.8 days) as did CTLA4Ig (MST: 21.5 +/- 1.7 days), but all grafts were rejected within 24 days. Importantly, combined blockade further and significantly prolonged allograft survival (MST: 75.3 +/- 12.7 days) and prevented the expansion and/or persistence of primed/effector alloreactive T cells. Our data suggest that CD134-CD134L is a critical pathway in alloimmune responses, especially recall/primed responses, and is synergistic with CD28-B7 in mediating T cell effector responses during allograft rejection. Understanding the mechanisms of collaboration between these different pathways is important for the development of novel strategies to promote long-term allograft survival.     

Expression of retrovirus-related, cytotoxic T lymphocyte- and transplantation-defined antigens in NIH/3T3 transfectants after a single passage in nude mice

Transfection of T24c-Ha-ras oncogene into NIH/3T3 fibroblasts resulted in the establishment of a transformed cell line (pT) that was tumorigenic when injected s.c. both into Swiss outbred nude mice and normal NIH inbred mice. The passage into nude mice, however, led to the development of a tumor variant (pT-nude) able to subsequently grow into sublethally x-irradiated but not into immunocompetent NIH mice. NIH mice immunized with this tumor variant developed a strong specific CTL response against the immunizing cell line, whereas the parental transformed pT cell line was not lysed. Clones were derived by limiting dilution from anti-pT-nude bulk population and were tested on a panel of transformed NIH/3T3 lines before and after their growth as tumor into nude mice. All of these lines were lysed by the Lyt-2+ CTL clones as a sole consequence of one in vivo passage into nude mice. The cross-reactive Ag were shown to be related to endogenous retroviral products as assessed by 1) immunoprecipitations of gp70, p15E, and p30 viral proteins in the nude variants but not in parental lines, and 2) by the ability of retroviruses from irradiated pT-nude cells to infect NIH/3T3 or pT lines making them susceptible to lysis by anti-pT-nude CTL clones. These results show that a single passage in nude mice can induce retrovirus-related, cell-surface Ag in transplanted neoplastic cells.     

The production of topoisomerase II-mediated DNA cleavage in human leukemia cells predicts their susceptibility to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)

Protein-associated DNA cleavage is produced in mammalian cells treated with active antileukemic DNA intercalating agents such as 4'(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). We have examined the ability of m-AMSA to produce DNA cleavage in 3 human myeloid leukemic cell lines with different sensitivities to the cytotoxic actions of m-AMSA to see if the magnitude of DNA cleavage correlated with the degree of m-AMSA sensitivity. DNA alkaline elution was used to quantify DNA cleavage. The amount of m-AMSA-induced DNA cleavage in the two lines sensitive to m-AMSA was 1-2 orders of magnitude greater than that in an m-AMSA-resistant leukemic line. The m-AMSA resistant line had been developed by prolonged exposure of one of the sensitive lines to m-AMSA. This finding was not secondary to a decreased uptake of m-AMSA in the resistant cell line. m-AMSA treatment of the nuclei isolated from the three lines produced DNA cleavage frequencies comparable to the cleavage frequencies produced by m-AMSA treatment of the whole cells from which the nuclei were isolated. The DNA cleaving ability stimulated by m-AMSA is thought to be mediated by drug-induced effects on topoisomerase II, a nuclear enzyme that mediates alterations in DNA conformation. Alterations in the manner in which this enzyme interacts with antineoplastic agents may explain the emergence of resistant cells following initially successful chemotherapy.     

Pterostilbene and 3'-hydroxypterostilbene are effective apoptosis-inducing agents in MDR and BCR-ABL-expressing leukemia cells

Pterostilbene and 3,5-hydroxypterostilbene are the natural 3,5-dimethoxy analogs of trans-resveratrol and piceatannol, two compounds which can induce apoptosis in tumor cells. In previous studies we demonstrated the importance of a 3,5-dimethoxy motif in conferring pro-apoptotic activity to stilbene based compounds so we now wanted to evaluate the ability of pterostilbene and 3,5-hydroxypterostilbene in inducing apoptosis in sensitive and resistant leukemia cells. When tested in sensitive cell lines, HL60 and HUT78, 3'-hydroxypterostilbene was 50-97 times more potent than trans-resveratrol in inducing apoptosis, while pterostilbene appeared barely active. However, both compounds, but not trans-resveratrol and piceatannol, were able to induce apoptosis in the two Fas-ligand resistant lymphoma cell lines, HUT78B1 and HUT78B3, and the multi drug-resistant leukemia cell lines HL60-R and K562-ADR (a Bcr-Abl-expressing cell line resistant to imatinib mesylate). Of note, pterostilbene-induced apoptosis was not inhibited by the pancaspase-inhibitor Z-VAD-fmk, suggesting that this compound acts through a caspase-independent pathway. On the contrary, 3'-hydroxypterostilbene seemed to trigger apoptosis through the intrinsic apoptotic pathway: indeed, it caused a marked disruption of the mitochondrial membrane potential delta psi and its apoptotic effects were inhibited by Z-VAD-fmk and the caspase-9-inhibitor Z-LEHD-fmk. Moreover, pterostilbene and 3'-hydroxypterostilbene, when used at concentrations that elicit significant apoptotic effects in tumor cell lines, did not show any cytotoxicity in normal hemopoietic stem cells. In conclusion, our data show that pterostilbene and particularly 3'-hydroxypterostilbene are interesting antitumor natural compounds that may be useful in the treatment of resistant hematological malignancies, including imatinib, non-responsive neoplasms.     

Gene therapy of rat malignant gliomas using neural stem cells expressing IL-12

Primary malignant brain tumors have a poor prognosis. This report investigates the potential for gene therapy of experimental brain tumors using neural stem cells (NSCs) expressing IL-12. In this study NSCs were isolated from the hippocampi of 3-5-month human embryos and used for lipofectamine mediated transfer of the IL-12 gene. Positive clones of anti-G418 were obtained and were proliferated in culture and expression of IL-12 was demonstrated by RT-PCR. For the in vivo studies three groups of rats were used and stereotactic injections were made into the striatum. In the first group C6 tumor cells were injected, in the second C6 cells and hNSCs. IL-12, and in the third C6 cells on Day 0 followed by hNSCs.IL-12 on day 5. The growth of the resulting tumors was monitored by magnetic resonance imaging (MRI) and after sacrifice by immunohistochemistry. Rats injected with C6 cells and hNSCs.IL-12 had a significantly prolonged survival. Injections of hNSCs.IL-12 were also made into established gliomas. The survival time was also significantly prolonged compared to controls. MR imaging demonstrated that there was initial growth of tumor followed by shrinkage and then disappearance. After sacrifice, tumor areas were studied by histochemistry. NSCs were often seen intermingled with tumor cells, particularly when they had been injected into established tumors; they were also present at the boundaries of the tumor mass. The immunohistochemical analysis showed that these infiltrates were mostly constituted by CD4(+) and CD8(+) T-lymphocytes, the CD8(+) being more numerous than the CD4(+). These findings indicated that NSCs engineered to release IL-12 could have a strong antitumor effect. Neural stem/precursor cells could be useful vectors in genetic approaches to brain tumors.     

Tumorigenesis by human herpesvirus 8 vGPCR is accelerated by human immunodeficiency virus type 1 Tat

Human herpesvirus 8 (HHV-8), also called Kaposi's sarcoma (KS) herpesvirus, can cause KS but is inefficient. Untreated human immunodeficiency virus type 1 (HIV-1) coinfection is a powerful risk factor. The HHV-8 chemokine receptor, vGPCR (ORF74), activates NF-kappaB and NF-AT, and their levels of activation are synergistically increased by HIV-1 Tat. Transgenic vGPCR mice develop KS-like tumors. A cell line derived from one such tumor expresses vGPCR and forms tumors in nude mice. Here we show that transfection of DNA encoding HIV-1 tat (but not a transactivation-defective mutant) into these tumor cells increases NF-kappaB and NF-AT activation levels and accelerates tumor formation. Tumorigenesis was also accelerated when Tat DNA was transfected into normal cells and the transfected cells were mixed with the tumor cells and injected into a single site. Tumorigenesis was also increased when the two cell types were injected at separate sites, suggesting that tumorigenesis is accelerated by Tat through soluble factors.