Tumor burden and clonality in multiple intestinal neoplasia mouse/normal mouse aggregation chimeras

Aggregation chimeras were formed between C57BL/6 mice heterozygous for the Apcmin (Min) mutation and wild-type SWR mice, that differ in their Pla2g2a status, a modifier of Apcmin, and also in their resistance to intestinal polyp formation. Variation in the dolichos biflorus agglutinin-staining patterns of the intestines of these mouse strains was used to determine the chimeric composition of the intestine in individual mice and to examine the clonal composition of adenomas. Macroscopic adenoma numbers in chimeric mice were compared with the expected adenoma numbers based on the percentage of C57BL/6J-Apcmin/+ epithelium in individual mice. These results unexpectedly show that there was no apparent inhibitory effect of the SWR-derived (Pla2g2a wild-type) tissue on adenoma formation in the C57BL/6J-Apcmin/+ epithelium. This suggests that the main genetic modifiers of the Min phenotype act at a cellular or crypt-restricted level with no discernable systemic effect. All adenomas were seen to contain C57BL/6J-Apcmin/+-derived epithelium, confirming that the germ-line mutation of the mApc gene is necessary to initiate tumorigenesis in this model system, and that the mApc gene acts in a cell autonomous fashion.

Defective placental vasculogenesis causes embryonic lethality in VHL-deficient mice

Inheritance of an inactivated form of the VHL tumor suppressor gene predisposes patients to develop von Hippel–Lindau disease, and somatic VHL inactivation is an early genetic event leading to the development of sporadic renal cell carcinoma. The VHL gene was disrupted by targeted homologous recombination in murine embryonic stem cells, and a mouse line containing an inactivated VHL allele was generated. While heterozygous VHL (+/−) mice appeared phenotypically normal, VHL −/− mice died in utero at 10.5 to 12.5 days of gestation (E10.5 to E12.5). Homozygous VHL −/− embryos appeared to develop normally until E9.5 to E10.5, when placental dysgenesis developed. Embryonic vasculogenesis of the placenta failed to occur in VHL −/− mice, and hemorrhagic lesions developed in the placenta. Subsequent hemorrhage in VHL −/− embryos caused necrosis and death. These results indicate that VHL expression is critical for normal extraembryonic vascular development.

Mouse mammary tumor virus/v-Ha-ras transgene-induced mammary tumors exhibit strain-specific allelic loss on mouse chromosome 4

Hybrid mice carrying oncogenic transgenes afford powerful systems for investigating loss of heterozygosity (LOH) in tumors. Here, we apply this approach to a neoplasm of key importance in human medicine: mammary carcinoma. We performed a whole genome search for LOH using the mouse mammary tumor virus/v-Ha-ras mammary carcinoma model in female (FVB/N × Mus musculus castaneus)F1 mice. Mammary tumors developed as expected, as well as a few tumors of a second type (uterine leiomyosarcoma) not previously associated with this transgene. Genotyping of 94 anatomically independent tumors revealed high-frequency LOH (≈38%) for markers on chromosome 4. A marked allelic bias was observed, with M. musculus castaneus alleles almost exclusively being lost. No evidence of genomic imprinting effects was noted. These data point to the presence of a tumor suppressor gene(s) on mouse chromosome 4 involved in mammary carcinogenesis induced by mutant H-ras expression, and for which a significant functional difference may exist between the M. musculus castaneus and FVB/N alleles. Provisional subchromosomal localization of this gene, designated Loh-3, can be made to a distal segment having syntenic correspondence to human chromosome 1p; LOH in this latter region is observed in several human malignancies, including breast cancers. Evidence was also obtained for a possible second locus associated with LOH with less marked allele bias on proximal chromosome 4.

Abnormal development of secondary lymphoid tissues in lymphotoxin β-deficient mice

The tumor necrosis factor (TNF) family cytokines lymphotoxin (LT) α and LTβ form heterotrimers that are expressed on the surface of activated lymphocytes and natural killer cells; LTα homotrimers can be secreted as well. Mice with a disrupted LTα gene lack lymph nodes (LN), Peyer’s patches (PP), and follicular dendritic cell (FDC) networks and reveal profound defects of the splenic architecture. However, it is unclear which of these abnormalities is the result of the absence in LTα homotrimers or LTαβ heterotrimers. To distinguish between these two possibilities, a mouse strain deficient in LTβ was created employing Cre/loxP-mediated gene targeting. Mice deficient in LTβ reveal severe defects in organogenesis of the lymphoid system similar to those of LTα−/− mice, except that mesenteric and cervical LN are present in most LTβ-deficient mice. Both LTβ- and LTα-deficient mice show significant lymphocytosis in the circulation and peritoneal cavity and lymphocytic infiltrations in lungs and liver. After immunization, PNA-positive B cell clusters were detected in the splenic white pulp of LTβ-deficient mice, but FDC networks were severely underdeveloped. Collectively, these results indicate that LTα can signal independently from LTβ in the formation of PNA-positive foci in the spleen, and especially in the development of mesenteric and cervical LN.

Tumor necrosis factor α plays a central role in immune-mediated clearance of adenoviral vectors

Adenovirus (Ad) gene transfer vectors are rapidly cleared from infected hepatocytes in mice. To determine which effector mechanisms are responsible for elimination of the Ad vectors, we infected mice that were genetically compromised in immune effector pathways [perforin, Fas, or tumor necrosis factor α (TNF-α)] with the Ad vector, Ad5-chloramphenicol acetyl transferase (CAT). Mice were sacrificed at 7–60 days postinfection, and the levels of CAT expression in the liver determined by a quantitative enzymatic assay. When the livers of infected mice were harvested 28 days postinfection, the levels of CAT expression revealed that the effectors most important for the elimination of the Ad vector were TNF-α > Fas > perforin. TNF-α did not have a curative effect on infected hepatocytes, as the administration of TNF-α to infected severe combined immunodeficient mice or to infected cultures in vitro had no specific effect on virus persistence. However, TNF-α-deficient mice demonstrated a striking reduction in the leukocytic infiltration early on in the infection, suggesting that TNF-α deficiency resulted in impaired recruitment of inflammatory cells to the site of inflammation. In addition, the TNF-deficient mice had a significantly reduced humoral immune response to virus infection. These results demonstrate a dominant role of TNF-α in elimination of Ad gene transfer vectors. This result is particularly important because viral proteins that disable TNF-α function have been removed from most Ad vectors, rendering them highly susceptible to TNF-α-mediated elimination.

Induction of minisatellite mutation in NIH 3T3 cells by treatment with the tumor promoter okadaic acid

Okadaic acid (OA) is a strong tumor promoter of mouse skin carcinogenesis and also a potent inhibitor of serine/threonine protein phosphatases. OA induces various genetic alterations in cultured cells, such as diphtheria-toxin-resistance mutations, sister chromatid exchange, exclusion of exogenous transforming oncogenes, and gene amplification. The present study revealed that it caused minisatellite mutation (MSM) at a high frequency in NIH 3T3 cells, although no microsatellite mutation was found. Nine of 31 clones (29%) exhibited MSM after 6 days of OA treatment, as opposed to only 1 of 30 clones (3%) without OA exposure. Moreover, NIH 3T3 cells treated with OA acquired tumorigenicity in nude mice, giving rise to 7 tumors within 25 weeks in 20 sites where 3 × 106 cells were injected. In contrast, the same numbers of untreated cells gave rise to only one tumor, and the tumor grew much slower. All of three OA-induced tumors examined manifested the MSM. The findings thus point to a molecular mechanism by which OA could function as a tumor promoter, and also the biological relevance of the induction of MSM in the tumorigenic process by OA.

Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization

Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund’s adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund’s adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund’s adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.

Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial growth factor isoforms VEGF121 and VEGF165 but not VEGF189

The vascular endothelial growth factor (VEGF) has been shown to be a significant mediator of angiogenesis during a variety of normal and pathological processes, including tumor development. Human U87MG glioblastoma cells express the three VEGF isoforms: VEGF121, VEGF165, and VEGF189. Here, we have investigated whether these three isoforms have distinct roles in glioblastoma angiogenesis. Clones that overexpressed each isoform were derived and inoculated into mouse brains. Mice that received VEGF121- and VEGF165-overexpressing cells developed intracerebral hemorrhages after 60–90 hr. In contrast, mice implanted with VEGF189-overexpressing cells had only slightly larger tumors than those caused by parental cells and little evidence of hemorrhage at these early times after implantation, whereas, after longer periods of growth, enhanced angiogenicity and tumorigenicity were apparent. There was rapid blood vessel growth and breakdown around the tumors caused by cells overexpressing VEGF121 and VEGF165, whereas there was similar vascularization but no eruption in the vicinity of those tumors caused by cells overexpressing VEGF189, and none on the border of the tumors caused by the parental cells. Thus, by introducing VEGF-overexpressing glioblastoma cells into the brain, we have established a reproducible and predictable in vivo model of tumor-associated intracerebral hemorrhage caused by the enhanced expression of single molecular species. Such a model should be useful for uncovering the role of VEGF isoforms in the mechanisms of angiogenesis and for investigating intracerebral hemorrhage due to ischemic stroke or congenital malformations.

Chromosomal aberrations in PARP−/− mice: Genome stabilization in immortalized cells by reintroduction of poly(ADP-ribose) polymerase cDNA

Depletion of poly(ADP-ribose) polymerase (PARP) increases the frequency of recombination, gene amplification, sister chromatid exchanges, and micronuclei formation in cells exposed to genotoxic agents, implicating PARP in the maintenance of genomic stability. Flow cytometric analysis now has revealed an unstable tetraploid population in immortalized fibroblasts derived from PARP−/− mice. Comparative genomic hybridization detected partial chromosomal gains in 4C5-ter, 5F-ter, and 14A1-C1 in PARP−/−mice and immortalized PARP−/−fibroblasts. Neither the chromosomal gains nor the tetraploid population were apparent in PARP−/− cells stably transfected with PARP cDNA [PARP−/−(+PARP)], indicating negative selection of cells with these genetic aberrations after reintroduction of PARP cDNA. Although the tumor suppressor p53 was not detectable in PARP−/− cells, p53 expression was partially restored in PARP−/− (+PARP) cells. Loss of 14D3-ter that encompasses the tumor suppressor gene Rb-1 in PARP−/− mice was associated with a reduction in retinoblastoma(Rb) expression; increased expression of the oncogene Jun was correlated with a gain in 4C5-ter that harbors this oncogene. These results further implicate PARP in the maintenance of genomic stability and suggest that altered expression of p53, Rb, and Jun, as well as undoubtedly many other proteins may be a result of genomic instability associated with PARP deficiency.

Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1β converting enzyme cDNA

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1β converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-γ. First, we compared the levels of IFN-γ secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-γ production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-γ levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8+ T cell-mediated and partially IFN-γ-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8+ T cells and IFN-γ-dependent pathways.

Potent inhibition of tumor survival in vivo by β-lapachone plus taxol: Combining drugs imposes different artificial checkpoints

Ablation of tumor colonies was seen in a wide spectrum of human carcinoma cells in culture after treatment with the combination of β-lapachone and taxol, two low molecular mass compounds. They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergism is schedule dependent; namely, taxol must be added either simultaneously or after β-lapachone. This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. There is little host toxicity. Cells can commit to apoptosis at cell-cycle checkpoints, a mechanism that eliminates defective cells to ensure the integrity of the genome. We hypothesize that when cells are treated simultaneously with drugs activating more than one different cell-cycle checkpoint, the production of conflicting regulatory signaling molecules induces apoptosis in cancer cells. β-Lapachone causes cell-cycle delays in late G1 and S phase, and taxol arrests cells at G2/M. Cells treated with both drugs were delayed at multiple checkpoints before committing to apoptosis. Our findings suggest an avenue for developing anticancer therapy by exploiting apoptosis-prone “collisions” at cell-cycle checkpoints.

Peptide-specific cytotoxic T lymphocytes restricted by nonself major histocompatibility complex class I molecules: Reagents for tumor immunotherapy

Studies in melanoma patients have revealed that self proteins can function as targets for tumor-reactive cytotoxic T lymphocytes (CTL). One group of self proteins MAGE, BAGE, and GAGE are normally only expressed in testis and placenta, whilst another group of CTL recognized proteins are melanocyte-specific differentiation antigens. In this study we have investigated whether CTL can be raised against a ubiquitously expressed self protein, mdm-2, which is frequently overexpressed in tumors. The observation that T-cell tolerance is self major histocompatibility complex-restricted was exploited to generate CTL specific for an mdm-2 derived peptide presented by nonself major histocompatibility complex class I molecules. Thus, the allo-restricted T-cell repertoire of H-2d mice was used to isolate CTL specific for the mdm100 peptide presented by allogeneic H-2Kb class I molecules. In vitro, these CTL discriminated between transformed and normal cells, killing specifically Kb-positive melanoma and lymphoma tumors but not Kb-expressing dendritic cells. In vivo, the CTL showed antitumor activity and delayed the growth of melanoma as well as lymphoma tumors in H-2b recipient mice. These experiments show that it is possible to circumvent T-cell tolerance to ubiquitously expressed self antigens, and to target CTL responses against tumors expressing elevated levels of structurally unaltered proteins.

Estimating the probability of initiated cell death before tumor induction

The effects of cell toxicity are known to be inherent in carcinogenesis induced by radiation or chemical carcinogens. The event of cell death precludes tumor induction from occurring. A long standing problem is to estimate the proportion of initiated cells that die before tumor induction. No experimental techniques are currently available for directly gauging the rate of cell death over extended periods of time. The obstacle can be surmounted by newly developed theoretical methods of carcinogenesis modeling. In this paper, we apply such methods to published data on multiple lung tumors in mice receiving different schedules of urethane. Bioassays of this type play an important role in testing environmental chemicals for carcinogenic activity. Our estimates for urethane-induced carcinogenesis show that, unexpectedly, many initiated cells die early in the course of tumor promotion. We present numerical estimates for the probability of initiated cell death for different schedules (and doses) of urethane administration.

Prevention of lymphocyte cell death in sepsis improves survival in mice

Sepsis induces extensive lymphocyte apoptosis, a process which may be beneficial to host survival by down-regulating the inflammatory response or, alternatively, harmful by impairing host defenses. To determine the beneficial vs. adverse effects of lymphocyte apoptosis in sepsis, we blocked lymphocyte apoptosis either by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl) fluoromethyl ketone (z-VAD), a broad-spectrum caspase inhibitor, or by use of Bcl-2 Ig transgenic mice that selectively overexpress the antiapoptotic protein Bcl-2 in a lymphoid pattern. Both z-VAD and Bcl-2 prevented lymphocyte apoptosis and resulted in a marked improvement in survival. z-VAD did not decrease lymphocyte tumor necrosis factor-α production. Considered together, these two studies employing different methods of blocking lymphocyte apoptosis provide compelling evidence that immunodepression resulting from the loss of lymphocytes is a central pathogenic event in sepsis, and they challenge the current paradigm that regards sepsis as a disorder resulting from an uncontrolled inflammatory response. Caspase inhibitors may represent a treatment strategy in this highly lethal disorder.

Tumor cells expressing a retroviral envelope escape immune rejection in vivo

A model system for the in vivo control of tumor cell proliferation by the immune system has been used to assay for the possible immunosuppressive activity of retroviral proteins. Expression vectors for the entire or the transmembrane subunit of the Moloney murine leukemia virus envelope protein were constructed, as well as control vectors for irrelevant transmembrane proteins—or no protein. They were introduced either into MCA205 murine tumor cells, which do not proliferate upon s.c. injection into an allogeneic host, or into CL8.1 murine tumor cells, which overexpress class I antigens and are rejected in a syngeneic host. In both cases, expression of the complete envelope protein or of the transmembrane subunit resulted in tumor growth in vivo, with no effect of control vectors. Tumor cell growth results from inhibition of the host immune response, as the envelope-dependent effect was no more observed for MCA205 cells in syngeneic mice or for CL8.1 cells in x-irradiated mice. This inhibition is local because it is not observed at the level of control tumor cells injected contralaterally. These results suggest a noncanonical function of retroviral envelopes in the “penetrance” of viral infections, as well as a possible involvement of the envelope proteins of endogenous retroviruses in tumoral processes.