New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors.

We prepared retrovirus packaging cell lines containing gag-pol genes from spleen necrosis virus (expressed from a cytomegalovirus promoter and the simian virus 40 (SV40) polyadenylation sequences) and, on a separate vector, either the env gene from spleen necrosis virus (expressed from the Rous sarcoma virus promoter and the SV40 polyadenylation sequences) or the env gene from amphotropic murine leukemia virus (expressed from a cytomegalovirus promoter and the SV40 polyadenylation sequences). The nucleotide sequences in these packaging cell lines have almost no homology to the retrovirus vectors we used. Retrovirus vectors were produced from these new helper cell lines without any genetic interactions between the vectors and sequences in the helper cells and without transfer of the packaging sequences.     

Human cells infected with retrovirus vectors acquire an endogenous murine provirus.

A 5.2-kilobase mouse RNA is expressed in human cells following infection with recombinant retroviruses propagated in mouse NIH 3T3 cells as psi-2 pseudotypes. This RNA is transcribed from a defective mink cell focus-forming provirus and copackaged into virions and integrated into human target cell DNA at a frequency comparable to that of the recombinant retrovirus genome.     

Spontaneous fusion in vivo between normal host and tumor cells: possible contribution to tumor progression and metastasis studied with a lectin-resistant mutant tumor.

Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGAr) mutant of the highly metastatic MDAY-D2 DBA/2 mouse tumor, led to the emergence of WGA-sensitive (WGAs) revertants having higher ploidy levels at the site of inoculation as well as at distant visceral metastases. The results implied that MDW4 was nonmetastatic but progressed to become metastatic in vivo only after a cellular change took place which was accompanied by extinction of the WGAr phenotype and acquisition of a higher number of chromosomes. Results presented here provide strong and direct evidence for the underlying mechanism being spontaneous cell fusion in vivo between the MDW4 (WGAr) tumor cells and normal host cells, at least some of which are of bone marrow origin. Thus, growth of the H-2d MDW4 tumor cells in (C3H X DBA/2)F1 (H-2k X H-2d) or (C57BL/6 X DBA/2)F1 (H-2b X H-2d) mice led to the appearance of WGAs revertants bearing the H-2k or H-2b major histocompatibility complex antigens associated with the C3H or C57BL/6 parental strains, respectively. Similarly, WGAs revertants of MDW4 were found to express H-2k antigens after growth in CBA/HT6T6 (H-2k) leads to DBA/2 bone marrow radiation chimeras. Attempts to mimic the in vivo hybridization process were successful in that in vitro somatic cell fusion between an ouabain-resistant (OuaR), 6-thioguanine-resistant (Thgr) derivative of the MDW4 mutant and either normal bone marrow or spleen cells resulted in loss of the WGAr phenotype in the hybrids (thus showing its recessive character) and increased malignant properties in vivo. An analysis of spontaneous frequencies of re-expression of various drug resistance genetic markers in several hybrid metastatic cells was also consistent with chromosome segregation of the sensitive alleles. The results show that tumor progression and the emergence of metastatic cell variants could arise as a consequence of tumor X host cell fusion followed by chromosome segregation. We also discuss the possibility that this type of event may normally be a very rare one during the growth of tumors, the frequency of which can be artificially amplified by the use of certain classes of lectin-resistant mutants carrying particular cell surface alterations.     

Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells.

The activity of the Moloney murine leukemia virus promoter is restricted in mouse embryonic stem cells. Gene expression with retrovirus vectors can be achieved in these cells if internal promoters are used. To address the possible influence of the viral enhancer sequences on expression from the internal promoter, we have constructed high-titer, self-inactivating retrovirus vectors which delete viral regulatory sequences upon integration in the host genome. We show that deleting most of the viral enhancer sequences has no significant effect on viral titer. This enhancer deletion leads to either an increase or a decrease in the amount of RNA transcribed from the internal promoter, but no consistent change can be found with any type of vector. The same changes in expression from the internal promoter observed in embryonic stem cells are also observed in 3T3 fibroblast cells, in which the viral promoter is active. These results indicate that viral regulatory elements influence expression from an internal promoter independently of expression from the virus promoter.     

BRE enhances in vivo growth of tumor cells

Human BRE, a death receptor-associating intracellular protein, attenuates apoptotic response of human and mouse tumor cell lines to death receptor stimuli in vitro. In this report, we addressed whether the in vitro antiapoptotic effect of BRE could impact on tumor growth in vivo. We have shown that the mouse Lewis lung carcinoma D122 stable transfectants of human BRE expression vector developed into local tumor significantly faster than the stable transfectants of empty vector and parental D122, in both the syngeneic C57BL/6 host and nude mice. In vitro growth of the BRE stable transfectants was, however, not accelerated. No significant difference in metastasis between the transfectants and the parental D122 was detected. Thus, overexpression of BRE promotes local tumor growth but not metastasis. We conclude that the enhanced tumor growth is more likely due to the antiapoptotic activity of BRE than any direct effect of the protein on cell proliferation.     

Gene transfer in swine embryos by injection of cells infected with retrovirus vectors

Key components for gene transfer to swine embryos using an avian retrovirus are described. A replication-defective reticuloendotheliosis (REV) viral vector can infect and be expressed in pig embryo fibroblasts (PEF). Infection with a replication-competent vector (REV-A) indicates a presumptive block to viral replication in PEF. Swine embryos obtained at the morula stage can be cultured in vitro to the blastocyst stage, injected with retrovirus helper cells or quail embryo fibroblasts producing REV, and transferred to recipient swine with survival to at least 6 wk of gestation.     

Alloantigen-induced cytotoxicity against syngeneic tumor cells: analysis at the clonal level

It has been shown that peritoneal exudate cells (PEC) from BALB/c mice immunized with minor histocompatibility antigens presented by DBA/2 or B10.D2 spleen cells are capable of lysing syngeneic YC8 tumor cells in a 4-hr 51Cr-release assay. In this study, we employed limiting dilution analysis to determine the frequency of CTL precursors (CTL-P) reactive against both the specific DBA/2 (or P815) target and the syngeneic tumor YC8. The mean frequency of anti-DBA/2 CTL-P in PEC from BALB/c mice immunized with DBA/2 was 1/302. Between one-third and one-fifth of limiting dilution microcultures that exhibited lytic activity against DBA/2 lymphoblasts (or P815) were also able to lyse YC8. No lysis of YC8 was observed in the absence of a parallel lysis on DBA/2 lymphoblasts or P815 target cells. T cell clones, derived by micromanipulation from microcultures selected for cytotoxic activity against YC8 and/or P815, maintained either the specific anti-allogeneic or the doubly reactive ( antiallogeneic plus anti-syngeneic tumor) phenotype. Fourteen clones (six specific and eight doubly reactive) were tested for cytotoxic activity on a panel of target cells with different haplotypes. All showed H-2-restricted specificity for minor histocompatibility antigens shared by DBA/2 and B10.D2. The restriction element for some of the clones mapped in the K region of the H-2 complex, whereas for other clones the restriction element mapped in the D region; both K- and D-restricted clones were able to lyse YC8. When the clones that exhibited lysis on YC8 were tested on two other BALB/c tumor targets, LSTRA, a Moloney virus induced lymphoma, and RL male-1, a radiation induced lymphoma, two of seven were found to lyse all three syngeneic tumor targets equally well, but not syngeneic BALB/c blasts. These clones were functionally categorized as conventional CTL because they were unable to proliferate when cultured with antigen in the absence of exogenous lymphokines, and were unable to produce lymphokine with IL 2 activity when stimulated by the appropriate splenocytes. When tested in vivo in a Winn assay, a strong anti-tumor activity against YC8 was exerted by the anti-DBA/2 clones DY4 -3 and DY16 -3. These clones lysed both YC8 and the immunizing target cells in vitro. No in vivo effect in neutralizing YC8 tumor growth was observed with clone D2-1, a clone that lysed DBA/2 targets but not YC8 in vitro.     

Instability of large direct repeats in retrovirus vectors.

Retrovirus vectors were constructed with large (0.85- to 1.3-kilobase-pair) direct repeats in their genomes. Deletions involving the direct repeats occurred at a high frequency. Deletions occurred both when the direct repeats were in tandem and when they were separated by additional sequences. These deletions occurred during virus replication.     

Conjunction of tumor cells with lymphocytes: implications for tumor invasion and metastasis

Our previous studies have led to a novel hypothesis that tumor metastasis is triggered by aberrant lymphocyte infiltration that disrupts intercellular junctions and surface adhesion molecules and causes dissociation of tumor cells from the primary tumor core, allowing lymphocytes to conjoin with dissociated tumor cells and physically 'drag' them to different tissue sites. Our hypothesis is supported by morphological and immunohistochemical data from multiple types of human cancer. This hypothesis challenges the traditional belief that the physical conjunction between tumor cells and lymphocytes would lead to degeneration of the tumor cells. To validate our hypothesis, H&E and immunostained sections were examined under high magnification to identify potential signs of degeneration-related changes. Our study revealed that >60% of isolated tumor cells overlying focal capsule disruptions, or within the stroma and vascular structures, were physically conjoined with lymphocytes to form tumor cell-lymphocyte chimeras (TLCs). Approximately 90% of the tumor cell partners of TLCs were morphologically indistinguishable from their counterparts within the tumor core. In addition, one third of the tumor cells of TLCs expressed high levels of cell proliferation specific proteins, or were undergoing mitosis. Our study also revealed that a subset of dilated lymphatic ducts or blood vessels at the site of focal capsule disruptions harbored variable numbers of tumor cells, and the wall of these structures was in direct physical continuity with the myoepithelial cell layer. Our study suggests that the onset of tumor metastasis may occur in two forms: (1) lymphocyte-mediated shuttling that allows lymphocytes to physically 'drag' tumor cells to different sites, and (2) tumor progenitor-mediated angiogenesis that allows tumor cells to directly enter the vascular structures.     

Nonspecific inhibition of tumor growth in vivo by admixed allogeneic tumor-sensitized lymphoid cells and identical inactivated allogeneic tumor cells.

With the in vivo tumor neutralization test (Winn test), growth of a transplanted (KMT-17) from Wistar-King-Aptekman rats was inhibited by allogeneic tumor (AH-66 from Donryu rats)-sensitized syngeneic lymphoid cells admixed with mitomycin C (MMC)-treated AH-66 cells. The observed tumor inhibition may be immunologically nonspecific, since no cross-antigens were detected by membrane immunofluorescence on the surfaces of KMT-17 and AH-66 cells. Close contact among KMT-17, AH-66-sensitized lymphoid cells and MMC-treated AH-66 cells was required for the inhibition of KMT-17 growth. AH-66 cells pretreated with formalin or ultrasonication lost tumor inhibitory activity when they were admixed with AH-66-sensitized lymphoid cells, and only MMC-treatment effectively preserved the tumor inhibitory activity of AH-66 cells. The sensitized spleen cells, draining lymph node, or peripheral blood cells inhibited tumor growth when they were admixed with MMC-treated AH-66 cells, whereas nucleated cells from bone marrow, thymus, or distal lymph node did not. Growths of KMT-17 were inhibited by admixed sensitized spleen cells and MMC-treated AH-66 even when pre-irradiated rats were used as recipients.     

Comparison of promoter suppression in avian and murine retrovirus vectors.

Previously, we described "promoter suppression" in infectious retrovirus vectors with two genes and an internal promoter. Here, we examined several parameters of promoter suppression and found that the amount of suppression in an integrated retrovirus vector was dependent both on whether the vector was derived from spleen necrosis virus or murine leukemia virus and on which internal promoter was present in the vector. Murine leukemia virus vectors showed less suppression than analogous spleen necrosis virus vectors. Furthermore, the amount of suppression was not dependent on either the relative strengths of the promoters nor the distance between the promoters. Moreover, we found that in vectors in which one promoter was suppressed, there was an inverse correlation between the DNaseI sensitivity of the chromatin surrounding a promoter and the suppression of its expression.     

Interleukin-2 inhibits proliferation of HPV-associated tumor cells and halts tumor growth in vivo

Previous studies have shown inhibition of cervical cancer cell growth by treatment with high concentrations of IL-2. In the present study, we evaluated the in vitro and in vivo effects of recombinant human IL-2 on HPV-associated tumor cells (3T3-16). Treatment of 3T3-16 cells with rhIL-2 for 72 h inhibited cell growth in a dose-dependent manner and this effect was evidenced at nanomolar concentrations. These tumor cells expressed mRNA for beta and gamma subunits of the IL-2 receptor, which are required for signal transduction. In experiments to explore the effect of IL-2 on the growth of the HPV-associated tumor, mice received rhIL-2 through different routes: (i) intraperitoneal; (ii) subcutaneous, at the tumor inoculation site; or (iii) subcutaneous, distant from the tumor inoculation site. An effective antitumor response was observed only in those animals that received IL-2 at the tumor site (P<0.01). These results indicate the potential adequacy of therapeutic strategies based on local administration of rhIL-2 for cervical carcinoma, not only based on the ability of this cytokine to stimulate cellular-mediated immunity but also because of its direct effects on tumor cells.     

Polyphenolics from peach (Prunus persica var. Rich Lady) inhibit tumor growth and metastasis of MDA-MB-435 breast cancer cells in vivo

The tumor growth inhibition and anti-metastatic effects of peach polyphenolics were investigated in vivo using a xenograft model and MDA-MB-435 breast cancer cells. Results showed that tumor growth and lung metastasis were inhibited in vivo by peach polyphenolics in a dose range of 0.8–1.6 mg/day, and these effects were mediated by inhibition of metalloproteinases gene expression. Modulation of metalloproteinase-2, metalloproteinase-3 and metalloproteinase-13 gene expression may be some of the molecular targets for anti-metastatic activity of peach polyphenolics. Therefore, these compounds may constitute a novel chemopreventive tool to reduce the risk of metastasis in the combination therapy when primary cancer is diagnosed. Conversion to equivalent human intake for future clinical studies using the body surface area normalization method gave a dose of ~370.6 mg/day for a human adult of 60 kg, which can be supplied by consuming two to three peach fruit per day or alternatively using a dietary supplement peach polyphenol extract powder.     

Co-inoculation of prostate cancer cells with U937 enhances tumor growth and angiogenesis in vivo

Tumor-associated macrophages (TAMs) have been implicated in promoting tumor growth and development. Here we present evidence that demonstrates that co-inoculation of male athymic nude mice with PC-3 prostate cancer cells and U937 promonocytic cells enhances tumor growth and increases tumor angiogenesis. Male athymic nude mice were co-inoculated with PC-3 and U937 cells (control or IL-4 stimulated) and tumor growth was monitored over time. Immunohistochemical analysis of tumor specimens was performed for proliferation markers (e.g., Ki67) and the effects of IL-4 stimulation on U937 cells were analyzed for chemokine expression. The presence of U937 cells increased the rate of tumor growth in vivo and stimulated increased microvascular density within the tumor bed. Stimulation of U937 cells with IL-4 resulted in a significant increase in several pro-angiogenic and pro-tumor chemokines (e.g., CCL2). Co-inoculation increases prostate cancer growth via upregulation of chemokines that induce angiogenesis within the tumor.