Multiple mutations are common at mouse Aprt in genotoxin-exposed mismatch repair deficient cells

Mismatch repair deficiency is known to contribute to elevated rates of mutations, particularly at mono- and dinucleotide repeat sequences. However, such repeats are often missing from the coding regions of endogenous genes. To determine the types of mutations that can occur within an endogenous gene lacking highly susceptible repeat sequences, we examined mutagenic events at the 2.3 kb mouse Aprt gene in kidney cell lines derived from mice deficient for the PMS2 and MLH1 mismatch repair proteins. The Aprt mutation rate was increased 33-fold and 3.6-20-fold for Mlh1 and Pms2 null cell lines, respectively, when compared with a wild-type kidney cell line. For the Pms2 null cells this increase resulted from both intragenic events, which were predominantly base-pairs substitutions, and loss of heterozygosity events. Almost all mutations in the Mlh1 null cells were due to base-pair substitutions. A:T-->G:C transitions (54% of small events) were predominant in the Pms2 null cells whereas G:C-->A:T transitions (36%) were the most common base-pair change in the Mlh1 null cells. Interestingly, 4-9% of the spontaneous mutant alleles in the mismatch repair deficient cells exhibited two well-separated base-pair substitution events. The percentage of mutant alleles with two and occasionally three base-pair substitutions increased when the Pms2 and Mlh1 null cells were treated with ultraviolet radiation (15-21%) and when the Mlh1 null cells were treated with hydrogen peroxide (35%). In most cases the distance separating the multiple base-pair substitutions on a given allele was in excess of 100 base-pairs, suggesting that the two mutational events were not linked directly to a single DNA lesion. The significance of these results is discussed with regards to the roles for the PMS2 and MLH1 proteins in preventing spontaneous and genotoxin-related mutations.     

Dose and frequency effect in mouse skin tumor promotion

In order to study the influence of both dose and application frequency of tumor-promoting agents on tumor development, we conducted a large-scale mouse skin two-stage carcinogenesis experiment. The back skins of 1110 CD-1 mice were painted once with 50 micrograms benzo(a)pyrene. These mice were divided into 24 groups according to subsequent schedules of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Mice were treated with TPA at five different frequencies, i.e., daily, or every second, 4th, 8th, or 16th day, and six different TPA doses per application were used (0.1, 0.2, 0.4, 0.8, 1.6, or 3.2 micrograms), which allowed groups to be established with the same total dose of TPA applied per time unit. Six of the 30 frequency/dose combinations at extreme low or high frequency and dose were excluded. At each fixed frequency of TPA application, there was a good dose-response of TPA in mouse skin papilloma incidence. There was also a good application frequency-response relationship at fixed doses of TPA application. Within the set of groups in which animals received the same total dose of TPA per time unit, some variation was observed with respect to frequency of application. In general, TPA applications every 4th and 8th day tended to yield a small number of tumors.     

Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells

The clinicopathological heterogeneity of glioblastoma (GBM) and the various genetic and phenotypic subtypes in GBM stem cells (GSCs) are well described. However, the relationship between GSCs and the corresponding primary tumor from which they were isolated is poorly understood. We have established GSC-enriched neurosphere cultures from 15 newly diagnosed GBM specimens and examined the relationship between the histopathological and genomic features of GSC-derived orthotopic xenografts and those of the respective patient tumors. GSC-initiated xenografts recapitulate the distinctive cytological hallmarks and diverse histological variants associated with the corresponding patient GBM, including giant cell and gemistocytic GBM, and primitive neuroectodermal tumor (PNET)-like components. This indicates that GSCs generate tumors that preserve patient-specific disease phenotypes. The majority of GSC-derived intracerebral xenografts (11 of 15) demonstrated a highly invasive behavior crossing the midline, whereas the remainder formed discrete nodular and vascular masses. In some cases, GSC invasiveness correlated with preoperative MRI, but not with the status of PI3-kinase/Akt pathways or O(6)-methylguanine methyltransferase expression. Genome-wide screening by array comparative genomic hybridization and fluorescence in situ hybridization revealed that GSCs harbor unique genetic copy number aberrations. GSCs acquiring amplifications of the myc family genes represent only a minority of tumor cells within the original patient tumors. Thus, GSCs are a genetically distinct subpopulation of neoplastic cells within a GBM. These studies highlight the value of GSCs for preclinical modeling of clinically relevant, patient-specific GBM and, thus, pave the way for testing novel anti-GSC/GBM agents for personalized therapy.     

Preexisting lymphatic endothelium but not endothelial progenitor cells are essential for tumor lymphangiogenesis and lymphatic metastasis

Endothelial progenitor cells have been shown to contribute to angiogenesis in various tumor models. Here, we have studied the relative contributions of bone marrow (BM)-derived endothelial progenitors and pre-existing lymphatic vessels to tumor lymphangiogenesis. We did not find significant incorporation of genetically marked BM-derived cells in lymphatic vessels during tumor- or vascular endothelial growth factor C-induced lymphangiogenesis. The degree of tumor lymphangiogenesis correlated with lymphatic vessel density in the peritumoral area, and despite tumor lymphangiogenesis, lymphatic metastasis failed to occur in gene-targeted vascular endothelial growth factor C(+/-) mice that have hypoplasia of the lymphatic network. Our data demonstrate that during tumor lymphangiogenesis and cancer cell dissemination via the lymphatics, the newly formed lymphatic vessels sprout from the pre-existing local lymphatic network with little if any incorporation of BM-derived endothelial progenitor cells.     

Sarcoma-180 cells and human colorectal tumor cells under in vitro hypoxic conditions are more sensitive to mitomycin C and carboquone

The effects of oxygen concentration on the chemosensitivity of mouse sarcoma-180 (S-180) cells and human colorectal cancer tissues to mitomycin C (MMC) or carboquone (CQ) were determined in vitro, since evidence had been obtained that these drugs are more effective in HeLa cells. The results were as follows: (a) S-180 cells exposed to various concentrations of MMC or CQ for 2 h under conditions of normal aeration (about 20%) or hypoxia (5.0% and 0%) were then maintained under normal conditions of aeration for 3 days. Change of viability was assessed by succinate dehydrogenase (SD) activity. With exposure of the cells to MMC or CQ, under anoxic conditions (O2:0%), SD activity decreased to a greater extent than seen in the control cells. The value for CQ was from 61.5% to about 36.2%. (b) The decrease in the SD activity of 20 colorectal cancer tissues kept under conditions of anoxia was compared with findings under normally aerated conditions, following exposure to 30 microM of MMC or 1.6 microM of CQ. On exposure to MMC or CQ under anoxic conditions, SD activity decreased significantly, compared with normally aerated conditions (P less than 0.001 for MMC; P less than 0.05 for CQ). As colorectal cancer is less sensitive than other tissues to various chemotherapeutic agents, we recommend that MMC and CQ be prescribed to treat patients with these malignant lesions.     

Microcinematographic study on the effect of methotrexate upon mouse mammary tumor cells (MMT cell line)

Summary The effect of methotrexate (amethopterin) upon the MMT cell line was studied by time-lapse microcinematography, the plasma levels obtained after systemic administration of maximum tolerated doses of the drug in man being simulated in vitro. Cells in the logarithmic growth phase (large growth fraction population) were widely affected, although enough drug-resistant cells remained to regenerate the cell colony. Cells in the preconfluent growth phase (small growth fraction population) were less effected, because many cells were arrested at the G₀-phase, ouside the cell cycle. A drug-resistant colony always developed, making the drug therapy useless. The experiments showed that rescue treatment with leucovorin (citrovorum factor of folinic acid) was not effective either, because, at least on our experimental conditions, recovery of the mitotic activity was more rapid and the number of degenerating cells smaller with rescue treatment than with the conventional treatment. The results also suggested a new mechanism of methotrexate action in addition to the classic one of folic acid inhibition, which might consist in the inhibition of the production of formyl-methionyl-tRNA, part of the initiation complex in protein biosynthesis.Coordinated center of the Consejo Superior de Investigaciones Cientificas, Madrid     

CIK细胞及其在肿瘤治疗中的应用

细胞因子诱导的杀伤(CIK)细胞过继免疫治疗在实体肿瘤治疗中发挥重要作用,特别是对于经标准治疗后出现复发或转移的实体肿瘤来说具有广阔的应用前景.尽管许多过继免疫治疗策略已经在临床前研究中取得了令人鼓舞的结果,但在临床推广应用中仍存在一些困难,如何确保抗肿瘤免疫效应物可以大量扩增并能在体内长时间停留,如何选择性杀伤肿瘤细胞以及确保应用安全等一系列关键问题仍亟待解决.     

Specific triiodothyronine binding by tumor cells and spleen cells in a thyroid hormone dependent mouse tumor system

We have reported thyroid hormone dependency of syngeneic mouse tumor (fibrosarcoma T241 in C57Bl/6J mice) for growth and spread. To study further the mechanisms of thyroid hormone action we assayed tumor cells and spleen cells for thyroid hormone receptors. Cells were incubated at 37°C for 1 hr with [¹²⁵I]triiodothyronine with and without unlabeled T3. Competitive inhibition of [¹²⁵I]T3 bound to spleen cells was demonstrated by increasing the amount of unlabeled T3 and T4. Similar competitive inhibition of binding was minimal for intact tumor cells. Saturable binding sites in muclei were demonstrated in all these cell populations. A Scatchard analysis of nuclei fraction from these cells showed equilibrium dissociation constant (K_d) of 0.56 × 10⁻⁹ for normal spleen cells and tumor cells and 1.4 × 10⁻⁹ M for tumor-bearing mice spleen cells. The estimated maximum binding capacity for nuclei of these cells was 26 fmol/10 × 10⁶ cells for normal spleen cells, 46 mol/10 × 10⁶ cells for tumor-bearing mice spleen cells and 45 fmol/10 #x0000D7; 10⁶ cells for tumor cells. Our results established the presence of T3 receptors in nuclei of tumor cells as well as spleen cells and suggests the direct metabolic effect of thyroid hormone as a possible mechanism for thyroid hormone dependency of this tumor.     

A role of interferon-gamma (IFN-gamma) in tumor immunity: T cells with the capacity to reject tumor cells are generated but fail to migrate to tumor sites in IFN-gamma-deficient mice

IFN-gamma-deficient (IFN-gamma-/-) mice induce potent in vitro immune responses such as anti-allo mixed lymphocyte reaction and CTL responses, whereas they often fail to exhibit in vivo immunity. Here, we investigated whether there exists a defect in tumor rejection responses and if so, which process of responses is impaired. IFN-gamma-/- and wild-type (WT) BALB/c mice were immunized with attenuated syngeneic CSA1M tumor cells. The capacity of T cells to mediate tumor protection was examined in Winn assays to assess the growth of tumor cells admixed with tumor-sensitized T cells. Splenic T cells from both groups of mice exhibited comparable levels of tumor-neutralizing activity. When portions of immunized mice were directly challenged with viable tumor cells, tumor rejection was induced only in WT mice. CD4(+) and CD8(+) T-cell infiltration were observed at the site of tumor challenge in WT mice, whereas such a T-cell infiltration did not occur in IFN-gamma-/- mice. Similarly, splenic T cells from interleukin 12-treated CSA1M-bearing IFN-gamma-/- and WT mice neutralized tumor cells at comparable efficacies in Winn assays. However, the migration of these T cells to tumor masses and the resultant interleukin 12-induced tumor regression took place in WT mice, but neither intratumoral T-cell infiltration nor tumor regression occurred in IFN-gamma-/- mice. These results indicate a critical requirement for IFN-gamma in the process of inducing T-cell migration to tumor sites rather than of generating antitumor protective T cells.     

Nitric oxide synthase is induced in tumor promoter-sensitive, but not tumor promoter-resistant, JB6 mouse epidermal cells cocultured with interferon-gamma-stimulated RAW 264.7 cells: the role of tumor necrosis factor-alpha

Expression of inducible nitric oxide synthase (iNOS) has been reported to be involved in certain organs of potential tumorigenesis, including the stomach and colon. The mechanisms for iNOS expression in epithelial cells, however, are not fully understood. In the present study, we investigated the role of macrophages in epithelial iNOS expression by coculturing a stimulated murine macrophage-like cell line, RAW 264.7, with either tumor promoter-sensitive (P+) or promoter-resistant (P-) JB6 murine epidermal cells. After monoculture, treatment of RAW 264.7 cells with IFN-gamma for 24 h generated a large amount of nitrite (NO2-), as reported previously, whereas no increase in NO2- concentration was observed in the IFN-gamma-treated P+ or P-subclones. Interestingly, when IFN-gamma-treated RAW 264.7 cells were cocultured with P+ but not P- cells, we observed a marked increase in NO2- concentration (30.8+/-3.6 microM), which significantly exceeded (P < 0.01) the sum of the concentrations (20.0+/-2.3 microM) added from each cell line monoculture. Western blotting analysis revealed that, after coculture, iNOS protein was up-regulated 55-fold more than the control in JB6 P+ but not in P- cells. IFN-gamma-treated RAW 264.7 cells secreted proinflammatory cytokines, including tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. The addition of IFN-gamma-treated RAW 264.7 cell-conditioned media to P+ subclones led to a significant enhancement of NO2- formation that was diminished by the TNF-alpha-specific but not IL-1beta-specific antibody. When combined with IFN-gamma, the recombinant TNF-alpha (1-100 ng/ml) enhanced NO2- formation in JB6 P+ cells, whereas IL-1beta (1-100 ng/ml) did not. These results led us to conclude that IFN-gamma-treated RAW 264.7 cells release TNF-alpha to induce iNOS expression in promoter-sensitive JB6 cells. Thus, we propose the hypothesis that macrophages stimulate neoplastic cells with TNF-alpha via a paracrine loop to induce epithelial iNOS protein expression.     

Microfluidic chip for isolation of viable circulating tumor cells of hepatocellular carcinoma for their culture and drug sensitivity assay

Circulating tumor cells (CTCs) have been proposed to be an active source of metastasis or recurrence of hepatocellular carcinoma (HCC). The enumeration and characterization of CTCs has important clinical significance in recurrence prediction and treatment monitoring in HCC patients. We previously developed a unique method to separate HCC CTCs based on the interaction of the asialoglycoprotein receptor (ASGPR) expressed on their membranes with its ligand. The current study applied the ligand-receptor binding assay to a CTC-chip in a microfluidic device. Efficient capture of HCC CTCs originates from the small dimensions of microfluidic channels and enhanced local topographic interactions between the microfluidic channel and extracellular extensions. With the optimized conditions, a capture yield reached > 85% for artificial CTC blood samples. Clinical utility of the system was further validated. CTCs were detected in all the examined 36 patients with HCC, with an average of 14 ± 10/2 mL. On the contrary, no CTCs were detected in healthy, benign liver disease or non-HCC cancer subjects. The current study also successfully demonstrated that the captured CTCs on our CTC-chip were readily released with ethylene diamine tetraacetic acid (EDTA); released CTCs remained alive and could be expanded to form a spheroid-like structure in a 3-dimensional cell culture assay; furthermore, sensitivity of released CTCs to chemotherapeutic agents (sorafenib or oxaliplatin) could be effectively tested utilizing this culture assay. In conclusion, the methodologies presented here offer great promise for accurate enumeration and easy release of captured CTCs, and released CTCs could be cultured for further functional studies.     

Nuclear accumulation of full-length and truncated adenomatous polyposis coli protein in tumor cells depends on proliferation

The adenomatous polyposis coli (APC) tumor suppressor is a nucleocytoplasmic protein. The nuclear accumulation of APC was recently found to vary depending on cell density, suggesting that putative APC function(s) in the nucleus is controlled by the establishment of cell contacts. We report here that the density-dependent redistribution of APC between nucleus and cytoplasm prevails in 6/6 thyroid and colorectal carcinoma cell lines. Moreover, mutated APC lacking known nuclear localization sequences had the similar distribution pattern as the full-length protein. APC invariably accumulated in the nuclei of Ki-67 expressing cells, but was largely cytoplasmic when cell cycle exit was induced by serum starvation or at high cell density. APC colocalized with beta-catenin in the nucleus only in one cell line (SW480). Also, APC maintained a predominantly nuclear position in early confluent states when cytoplasmic beta-catenin was recruited to newly formed adherens-like junctions. The results indicate that nuclear targeting of APC is driven by cell cycle entry rather than altered cell-cell contact. The ability of C-terminally truncated APC to accumulate in the nucleus suggests that nuclear import signals other than NLS1(APC) and NLS2(APC) are functionally important. Residual function(s) of N-terminal APC fragments in tumor cells carrying APC mutations might be beneficial to tumor growth and survival.     

Correlation of frequency of induced mutation and metastatic potential in tumor cell lines from a single mouse mammary tumor

Spontaneous mutation rates were determined in mouse mammary tumor subpopulation lines that differ in metastatic phenotype. Although there was almost a 9-fold difference in spontaneous rates to ouabain resistance among the three lines tested, the difference did not correlate with ability to metastasize. Similarly a 10-fold difference in spontaneous rates to 6-thioguanine resistance did not correlate with metastatic ability. In contrast, the frequency of ethyl methanesulfonate-induced mutations was associated with metastatic potential. Thus, ethyl methanesulfonate only induced significant numbers of 6-thioguanine resistant colonies in 66 and 410.4 cells, the only 2 of 5 lines tested that spontaneously metastasize at high frequency, and of ouabain resistant colonies in 66, 410.4, and 168 cells, the only lines tested that produce experimental lung metastases after i.v. injection. Differential sensitivity to induced mutation was not correlated with differences in plating efficiency, wild type sensitivity to ethyl methanesulfonate, 6-thioguanine, or ouabain toxicity, ploidy, cell shape, cell size, or ability to engage in metabolic cooperation.     

p53 transdominance but no gain of function in mouse brain tumor model

Although p53 mutations in tumors typically result in loss of transactivation of p53 target genes some mutants display gain-of-function activity. The latter has important implications for the design of rational cancer therapy. We previously described a germ-line p53 mutation (deletion of codon 236, Y236delta) associated with a familial brain tumor syndrome. To determine whether this tissue-specific tumor predisposition reflects a gain-of-function activity of Y236delta or an effect of genetic background we have developed a mouse brain tumor model. Primary neuroectodermal cells deficient for p53 (+/- or -/-) and transduced with Y236delta using a retroviral vector were transplanted into the brain of adult wild-type mice. This neurografting paradigm circumvents the problem of early lethal tumors at extracerebral sites associated with germ-line p53 deficiency. Brain tumors arising in this mouse model were highly invasive, reflecting an important feature of the human disease. Tumors arose from p53+/- cells only when transduced with Y236delta. In keeping with in vitro data showing that Y236delta has dominant-negative activity, these tumors retained the endogenous wild-type p53 allele but accumulated high levels of Y236delta. However, the presence of Y236delta in transplanted p53-/- cells had no effect on the tumor frequency, 15% versus 27% without the mutant. In conclusion, Y236delta is transdominant but exerts no gain-of-function activity mediating a more penetrant tumor phenotype.     

Lymphocytic infiltration and cytotoxicity under hypoxic conditions in the EMT6 mouse mammary tumor

Infiltration of lymphocytes, neutrophils and macrophages was evaluated in hypoxic and well-oxygenated areas of the EMT6 mouse mammary adenocarcinoma, by in vivo staining with the fluorescent dye Hoechst 33342 followed by cell sorting on the basis of fluorescence intensity. Tumors were grouped by days post-injection (days 11-14, 15-17 and 20-27). As lymphocytes are the only host cell population in this tumor model to possess lytic activity against EMT6 tumor cells, the ability of sensitized T lymphocytes to lyse syngeneic EMT6 cells was examined under conditions of varying oxygen concentrations. Infiltrating lymphocytes were detected to the same extent in cell fractions from both areas in all tumors. In contrast, neutrophils were found in significantly higher percentages in the hypoxic population than in the well-oxygenated cell fraction of all but the largest tumors. Macrophages were present in significantly higher percentages in the well-oxygenated fraction than in the hypoxic fraction of day-11 to -14 tumors. Extreme radiobiological hypoxia (0% O2) resulted in a significant decrease in T-cell-mediated lysis of EMT6 tumor cells, compared to lysis in room air (20% O2), but lysis was not impaired under conditions of mild radiobiological hypoxia (1% O2). Our study indicates that host-cell infiltration into areas of differing oxygenation may be quantitated via in situ Hoechst staining followed by cell sorting; in the EMT6 tumor, lymphocytes appear to infiltrate hypoxic areas to the same extent as well-oxygenated areas, and T-lymphocyte killing of syngeneic tumor cells is significantly reduced, although still present, under these hypoxic conditions.