Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes.

These studies have compared the ability of NIH 3T3 cells containing different ras oncogenes to form tumor nodules in the lungs of nude mice after tail vein injection. The genes studied include the normal cellular and bladder tumor ras genes, recombinant viral/cellular ras genes, recombinant yeast/mammalian ras genes, and a constructed gene with yeast RAS1 sequences significantly modified by deletions and an oncogenic mutation. The results show that NIH 3T3 cells containing these genes readily form lethal tumor nodules in the lungs of nude mice after tail vein injection. No control NIH 3T3 cells formed lung tumors within 66 days. Although there were some quantitative differences in the potencies of the various lines, the striking conclusion is that NIH 3T3 cells transformed by either normal or activated mammalian ras genes form approximately equal numbers of experimental lung metastases. In addition, cells transformed by a significantly modified yeast RAS1 gene containing a purposefully introduced oncogenic mutation were also equally active in this assay. The amount of p21 (the 21-kDa protein encoded by ras), as measured by immunoprecipitation, was approximately the same in the parent lines before injection as in the tumors recovered after injection. This result indicates that there is no selection for metastatic sublines containing larger quantities of p21. Transfection of EJ bladder tumor ras DNA into NIH 3T3 cells followed by injection 3 days later into the tail veins of nude/beige mice indicated that the EJ ras gene can confer a metastatic phenotype within 3.5 cell generations without selection or clonal growth in vitro. Thus, the biochemical changes initiated after introduction of the c-Ha-ras gene into NIH 3T3 cells result in the almost immediate acquisition of phenotypes necessary for experimental metastasis.     

Amplification and rearrangement of the Kirsten ras oncogene in virus-transformed BALB/c 3T3 cells during malignant tumor progression.

Analyses of the cellular and viral Kirsten ras genes (c-Ki-ras and v-Ki-ras, respectively) during malignant tumor progression were performed by using Kirsten murine sarcoma virus-transformed BALB/c 3T3 cells that harbor a replication-defective provirus. After injection into athymic nude mice by four different routes, primary tumors and secondary lung metastases were isolated, adapted to in vitro growth, and analyzed for DNA levels and mRNA expression of both genes for comparison with the originally injected transformed cells and untransformed 3T3 cells. For all tumors (primary or secondary), the v-Ki-ras gene was amplified and v-Ki-ras mRNA expression was highly elevated above that observed in the original transformed cell population. In two of five lung metastases from the i.v. and footpad injection routes, rearranged Ki-ras DNA sequences were observed. Micrometastases from the s.c. route of injection did not display these alterations. Injection of footpad lung tumor cells with rearrangements into a second group of animals led to multiple lung metastases with even further rearrangements correlating with more effective lung colonization/growth ability (overt lung tumors in five of eight animals less than 20 days after injection). However, reinjection of an i.v. lung tumor with rearranged Ki-ras led to no further rearrangements in the lung microfoci tumors isolated greater than 40 days after injection. These data suggest (i) the significance of amplification and elevated expression of v-Ki-ras in tumor formation, (ii) correlation of this amplification with more effective tumor progression, and (iii) the selective advantage that cells with Ki-ras DNA sequence additions have in the formation of overt lung tumors.     

Signaling pathways in Ras-mediated tumorigenicity and metastasis

The effector domain mutants of oncogenic Ras, V12S35 Ras, V12G37 Ras, and V12C40 Ras were tested for their abilities to mediate tumorigenic and metastatic phenotypes in athymic nude mice when expressed in NIH 3T3 fibroblasts. All mutants displayed comparable tumorigenic properties, but only the mutant that activates the Raf-mitogen-activated protein kinase kinase (MEK)-extracellular regulated kinase (ERK) 1/2 pathway, V12S35 Ras, induced tumors in the experimental metastasis assay. Furthermore, direct activation of the MEK-ERK1/2 pathway in NIH 3T3 cells by mos or a constitutively active form of MEK was sufficient to induce metastasis whereas R-Ras, which fails to activate the ERK1/2 pathway, is tumorigenic but nonmetastatic. The subcutaneous tumors and lung metastases derived from V12S35 Ras-transformed NIH 3T3 cells expressed higher levels of activated ERK1/2 in culture when compared with the parental cellular pool before injection, indicating that selection for cells with higher levels of activated ERK1/2 occurred during tumor growth and metastasis. By contrast, cells explanted from V12G37-Ras or V12C40-Ras-induced tumors did not show changes in the level of ERK1/2 activation when compared with the parental cells. When tumor-explanted cell lines derived from each of the effector domain mutants were passaged one additional time in vivo, all mediated rapid tumor growth, but, again, only cells derived from V12S35 Ras-tumors formed numerous metastatic lesions within the lung. These results show that the metastatic properties of the Ras effector domain mutants segregate, and that, whereas Ras-mediated tumorigenicity can arise independently of ERK1/2 activation, experimental metastasis appears to require constitutive activation of the ERK1/2 pathway.     

Amplification of the proto-neu oncogene facilitates oncogenic activation by a single point mutation.

To determine whether the amplification of the proto-neu oncogene (also called c-erbB-2) plays a role in tumorigenicity, we previously generated an NIH 3T3 transfectant (DHFR/G-8) that carried the amplified proto-neu gene. The DHFR/G-8 cells exhibited normal morphology. Their growth curve was similar to that of NIH 3T3 cells but was different from that of the B104-1 cell, and NIH 3T3 transfectant that carries the activated neu oncogene. When injected into nude mice, B104-1 cells produced tumors within 2 weeks, whereas the DHFR/G-8 cells did not produce tumors until 3 months after injection, and the NIH 3T3 cells did not produce any tumors even after 3 months. The tumors produced by the injection of the DHFR/G-8 cells were excised and grown in culture. The cells derived from the tumors were of transformed morphology and highly tumorigenic. The DNAs from the tumor cells were transfected into NIH 3T3 cells. The transfection resulted in foci on the NIH 3T3 monolayer. Southern analysis indicated that the foci derived from the transfection contained the neu gene. Using oligonucleotides as probes, the neu gene in the foci was found to carry a single-point mutation identical to the one previously found in the rat neuroblastoma and glioblastoma induced by the ethylnitrosourea. We conclude that the DNA region encoding the transmembrane domain of neu is a hot spot for converting the proto-neu gene into an activated oncogene and that amplification of the proto-neu gene facilitates mutation of the hot spot.     

Is tail vein injection a relevant breast cancer lung metastasis model?

Background

Two most commonly used animal models for studying breast cancer lung metastasis are: lung metastasis after orthotopic implantation of cells into the mammary gland, and lung implantations produced after tail vein (TV) injection of cells. Tail vein injection can produce lung lesions faster, but little has been studied regarding the differences between these tumors, thus, we examined their morphology and gene expression profiles.

Methods

Syngeneic murine mammary adenocarcinoma, 4T1-luc2 cells, were implanted either subcutaneously (Sq), orthotopically (OS), or injected via TV in Balb/c mice. Genome-wide microarray analyses of cultured 4T1 cells, Sq tumor, OS tumor, lung metastases after OS (LMet), and lung tumors after TV (TVt) were performed 10 days after implantation.

Results

Bioluminescence analysis demonstrated different morphology of metastases between LMet and TVt, confirmed by histology. Gene expression profile of cells were significantly different from tumors, OS, Sq, TVt or LMet (10,350 probe sets; FDR≤1%; P<0.0001). Sq tumors were significantly different than OS tumors (700 probe sets; FDR≤15%; P<0.01), and both tumor types (Sq and OS) were significantly different than LMet (1,247 probe sets; >1.5-fold-change; P<0.01), with no significant difference between TVt and LMet.

Conclusions

There were significant differences between the gene profiles of cells in culture and OS versus LMet, but there were no differences between LMet versus TVt. Therefore, the lung tumor generated by TVt can be considered genetically similar to those produced after OS, and thus TVt is a relevant model for breast cancer lung metastasis.KEY WORDS : Breast cancer, lung metastasis, animal model, microarray, metastasis model     

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.     

Novel experimental models of human cancer metastasis in nude mice: Lung metastasis,intraabdominal carcinomatosis with ascites,and liver metastasis

Summary The ability of RPMI4788 cells, a human colon cancer cell line, to produce experimental metastases in the lung, intraperitoneal cavity, and liver was studied in nude mice. Injection of 2×10⁶ tumor cells into the tail vein of nude mice produced metastatic lung tumors, and an intraportal injection of 5×10⁶ cells produced metastatic liver tumors. An intraabdominal carcinomatosis with ascites was formed after an i.p. injection of 5×10⁶ tumor cells. The nude mice with lung metastasis or intraabdominal carcinomatosis always died within a few weeks. Macroscopic observation showed that the number of lung metastatic nodules on day 21 after tumor inoculation was 311.3±78.2 (mean±SD) in BALB/C nude mice, and 187.5±26.7 in ICR nude mice. In survival experiments, the mice with intraabdominal carcinomatosis showed a mean survival of 29.0±1.7 (mean±SD) days in BALB/C nude mice and 43.6±6.1 days in ICR nude mice. These novel experimental models of metastases in nude mice produced by injection of RPMI4788 cells had high reproducibility and may be useful not only for the study of the metastatic process but also for testing anticancer drugs.     

Activated protooncogenes in human lung tumors from smokers.

Fourteen primary human lung tumor DNAs from smokers were analyzed for transforming activity by two DNA transfection assays. Activated protooncogenes were detected in 3 of 11 tumor DNAs by the NIH 3T3 focus assay, whereas activated protooncogenes were detected in 11 of 13 tumor DNAs by the NIH 3T3 cotransfection-nude mouse tumorigenicity assay. K- or NRAS genes activated by point mutation at codons 12 or 61 were detected in a large cell carcinoma, a squamous cell carcinoma, and 5 adenocarcinomas. An HRAS oncogene activated by a different mechanism was detected in an epidermoid carcinoma. One adenocarcinoma was found to contain an activated RAF gene. Two unidentified transforming genes were detected in a squamous cell carcinoma DNA and two adenocarcinoma DNAs. Eight of 10 lung adenocarcinomas that had formed metastases at the time of surgery were found to contain RAS oncogenes. No significant increase in metastasis was observed in the lung adenocarcinomas that contained one or more 6-kilobase EcoRI alleles of the LMYC gene. Overall, 12 of 14 (86%) of the lung tumor DNAs from smokers were found to contain activated protooncogenes. RAS oncogenes appear to play a role in the development of metastases in lung adenocarcinomas.     

Invasiveness and metastasis of NIH 3T3 cells induced by Met-hepatocyte growth factor/scatter factor autocrine stimulation.

The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). NIH 3T3 cells express HGF/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH cells are invasive in vitro and display enhanced protease activity necessary for the invasive phenotype. In experimental and spontaneous metastasis assays, Metmu or HMH cells metastasize to the lung, but lower numbers of subcutaneously injected Metmu and HMH cells produced invasive tumors in the heart, diaphragm, salivary gland, and retroperitoneum. It has been reported elsewhere that Met expression increased with tumor passage in athymic nude mice, and these tumor explants show enhanced activity in the metastasis assays. Autocrine-mediated Met-HGF/SF signal transduction in NIH 3T3 mesenchymal cells may provide an important system for understanding the biological process of metastasis.     

A pulmonary metastatic model of human non-small cell lung carcinoma cells that produce a neutrophil elastase-like molecule in severe combined immunodeficiency mice

STUDY OBJECTIVES: To establish a clinically relevant animal model of pulmonary metastases of human non-small cell lung carcinoma (NSCLC) cells in severe combined immunodeficiency (SCID) mice, which can be used for repetitive investigations, so as to improve our understanding and management of the cellular and molecular mechanisms of human lung cancer metastases. METHODS AND RESULTS: SCID mice subcutaneously injected in the flank with 1 x 10(6) EBC-1 cells derived from human lung squamous cell carcinoma were killed weekly for examination until 12 weeks after tumor inoculation. The biological characteristics of implanted tumors and their metastatic foci were investigated by hematoxylin-eosin staining and immunostaining for neutrophil elastase (NE). Three weeks after ectopic implantation, EBC-1 cell lines formed a tumor at the inoculation site and grew steadily to show a plateau at 10 weeks. EBC-1 cells formed multiple metastases in the lung 7 weeks after tumor inoculation; their numbers increased steadily until 12 weeks in all mice. Immunoreactivity for NE was intense in the metastatic tumor cells. Then, to establish the primary tumor amputation/pulmonary metastasis model and to evaluate how primary tumor amputation influences the development of pulmonary metastases at the cellular and molecular level, excision was performed before (3 weeks and 5 weeks after inoculation) and after (7 weeks and 9 weeks after inoculation) formation of lung metastases. When the primary tumor was excised 3 weeks after tumor inoculation, all mice had pulmonary metastasis at 12 weeks after inoculation. Blood samples obtained at 3 weeks after tumor inoculation contained human beta-actin messenger RNA, which represents circulating tumor cells. CONCLUSION: Our NSCLC EBC-1 pulmonary metastasis model is reliable, technically simple, and predictably results in pulmonary metastasis from early hematogenous spread. This model may be useful for elucidating the mechanism of pulmonary metastasis in human lung cancer, and testing anti-metastatic efficacy of therapeutic agents in vivo.     

Mina53, a novel c-Myc target gene, is frequently expressed in lung cancers and exerts oncogenic property in NIH/3T3 cells

Purpose

Mina53, whose expression is directly induced by c-Myc, is overexpressed in various cancers and plays an important role in cell growth. To clarify the involvement of Mina53 in lung cancers, we investigated its expression in human lung cancer tissues as well as in various lung cancer cell lines.

Methods

Mina53 expression was determined by real-time RT-PCR, western blotting, and immunohistochemistry using lung cancer cell lines, normal human bronchial epithelial cells, and lung cancer tissues. Biological effects of Mina53 were evaluated by soft agar colony formation assay and tumorigenicity in nude mice using Mina53-transfected NIH/3T3 cells. cDNA microarray analysis was performed to determine the gene alteration by Mina53 and confirmation was made using real-time RT-PCR with mina53 expression plasmid or mina53 shRNA-transfected NIH/3T3 cells.

Results

We observed that 62% of patients evidenced overexpression of Mina53 from the early clinical stages of lung cancer. Differences according to gender, smoking status, or histologic type were not statistically significant. Forced expression of Mina53 in NIH/3T3 cells induced cell transformation, and mina53-transfected NIH/3T3 clones produced tumors in nude mice, demonstrating that Mina53 has oncogenic potential. cDNA microarray revealed that 254 genes had altered expression in a mina53-transfected NIH/3T3 clone. Mina53 regulates several genes related to cell adhesion and metabolism, which have also been reported to be regulated by c-Myc. Genes regulated by Mina53, but not by c-Myc included cytokine/growth factor related genes such as EGFR, IL-6, and HGF.

Conclusion

Our results suggest that Mina53 plays an important role in carcinogenesis and may be a target for cancer prevention.     

Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase.

RNA-dependent protein kinase is a M(r) 68,000 protein in human cells (p68 kinase) or a M(r) 65,000 protein in murine cells (p65 kinase). p65/p68 is a serine/threonine kinase induced by interferon treatment and generally activated by double-stranded RNAs. Once activated, the known function of this kinase is inhibition of protein synthesis through phosphorylation of the eukaryotic initiation factor 2. Here we have investigated the potential for tumorigenicity in mice of murine NIH 3T3 clones expressing human p68 kinase, either the wild-type or a mutant inactive kinase with a single amino acid substitution in the invariant lysine-296 in the catalytic domain II. Expression of the mutant p68 kinase was correlated with a malignant transformation phenotype, giving rise to the production of large tumors of at least 1 cm in diameter within 7-12 days in all inoculated mice. In contrast, no tumor growth was observed for several weeks in mice inoculated with NIH 3T3 cell clones expressing either the wild-type recombinant p68 kinase or only the endogenous p65 kinase, the murine analogue of the p68 kinase. These results suggest that functional p65/p68 kinase (recently called PKR), by a still undefined mechanism, may also act as a tumor suppressor. Consequently, one of the pathways by which interferon inhibits tumor growth might be through its capacity to induce the enhanced expression of this kinase.     

CD3-mediated activation of tumor-reactive lymphocytes from patients with advanced cancer

Lymphocytes from blood or tumors of patients with advanced cancer did not proliferate and produced very low levels of tumor necrosis factor and IFN-gamma when cultured with autologous tumor cells. Proliferation and lymphokine production dramatically increased in the presence of beads conjugated with mAbs to CD3 plus mAbs to CD28 and/or CD40, and the lymphocytes destroyed the tumor cells. Expression density of CD3 concomitantly increased from low to normal levels. Furthermore, beads providing a CD3 signal (in combination with CD28 or CD28 plus CD40) gave partial protection against the inhibitory effect of transforming growth factor type beta1 on lymphocyte proliferation and production of tumor necrosis factor and IFN-gamma. MHC class I-restricted cytolytic T cells lysing autologous tumor cells in a 4-h Cr(51) release assay were generated when peripheral blood leukocytes were activated in the presence of autologous tumor cells and anti-CD3/CD28 or anti-CD3/CD28/CD40 beads. Experiments performed in a model system using anti-V-beta1 or anti-V-beta2 mAbs to activate subsets of T cells expressing restricted T cell receptor showed that lymphocytes previously activated by anti-V-beta can respond to CD3 stimulation with vigorous proliferation and lymphokine production while retaining their specificity, also in the presence of transforming growth factor type beta1. Our results suggest that T lymphocytes from cancer patients can proliferate and form Th1 type lymphokines in the presence of autologous tumor cell when properly activated, and that antigen released from killed tumor cells and presented by antigen-presenting cells in the cultures facilitates the selective expansion of tumor-directed, CD8(+) cytolytic T cells.     

Selection of liver-colonizing tumor cells from a murine fibrosarcoma induced by methylcholanthrene

Summary An experimental tumor model was developed to study the organ preference of malignant tumors. The primary tumor ER 15-P was induced by in-oculation of 1 mg methylcholanthrene in 0.1 ml sesame oil into the left femoral muscle of a female C57/B1₆J mouse. The tumor was palpable 100 days after induction. Spontaneous lung metastases were found at autopsy on day 128. Serial IM transplantation of tumor cells from the primary ER 15-P resulted in pulmonary metastases in all male and female mice. After IV injection of tumor cells from ER 15-P to male mice, colonies were found in lungs, thoracic cavity, liver, kidneys and occasionally also adrenals; female mice sometimes had ovarian metastases in addition, but no hepatic metastases. Liver-colonizing tumor cells were selected in male mice as follows: (a) IV injection of tumor cells from primary ER 15-P; (b) removal of tumor cells from liver tumor nodules, reinjection into mesenteric vein; (c) preparation of resulting tumors in the liver, reinjection of these cells through the portal system in one group of mice, and IV administration into tail vein in another group: (d) IM inoculation of tumor cells of the mesenteric passage in the left gastrocnemius muscle of mice prior to IV injection via tail vein in another group. Steps c and d were repeated three times. The procedure resulted in a highly significant decrease of tumor cell colonization to lungs and other organs, and a preferential increase of liver colonization. The liver preference of cell lines thus selected was obvious. Possible mechanisms for the organ preference of malignant tumors are discussed.     

Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F1 mouse.

Activated c-Ha-ras protooncogenes have recently been identified in the DNA of some spontaneous hepatic tumors found in 2-year-old B6C3 F1 mice. Activation of c-Ha-ras has now been demonstrated in DNA from well-differentiated hepatomas initiated by a single dose of carcinogen given to male B6C3 F1 mice at 12 days of age. DNA from each of 25 hepatomas, induced by N-hydroxy-2-acetylaminofluorene, vinyl carbamate, or 1'-hydroxy-2',3'-dehydroestragole, containing transforming activity in the NIH 3T3 transfection assay. Southern analysis of NIH 3T3 cells transformed by DNA from 24 of these hepatomas revealed amplified and/or rear-ranged restriction fragments homologous to a Ha-ras probe. The other tumor contained an activated Ki-ras gene. Immunoprecipitation and NaDodSO4/PAGE analysis of p21 ras proteins in NIH 3T3 transformants derived from a majority of the hepatomas suggested that the activating mutations were localized in the 61st codon of the c-Ha-ras gene. Creation of a new Xba I restriction site by an AT----TA transversion at the second position of codon 61 was detected in DNA from primary tumors and NIH 3T3 cells transformed by DNA from 6 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced hepatomas. Selective oligonucleotide hybridization demonstrated a CG----AT transversion at the first position of the 61st codon in NIH 3T3 transformants derived from 7 of 7 N-hydroxy-2-acetylaminofluorene-induced hepatomas. By the same criterion, an AT----GC transition at the second position of codon 61 was the activating mutation in 1 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced tumors. Thus, c-Ha-ras activation is apparently an early event in B6C3 F1 mouse hepatocarcinogenesis that results directly from reaction of ultimate chemical carcinogens with this gene in vivo.     

Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis-related genes through cDNA microarray

PURPOSE: To establish a hepatocellular carcinoma (HCC) cell line from lung metastatic lesions of human HCC in nude mice so as to provide a suitable model for the study of lung-metastasis-related molecular mechanisms. METHODS: HCC clone cells MHCC97-H were inoculated into BALB/c nude mice, and the pulmonary metastatic lesions were harvested and re-implanted into nude mice for the second round of in vivo selection. The same procedure was repeated twice. A new cell line from the third round of lung metastases was established. RESULTS: A human HCC cell line with unique metastatic characteristics was established by in vivo selection. This cell line, designated as HCCLM3, was polygonal epithelial cell with hypotriploid karyotype and population doubling time of 34.9 h. The cells were positive for alpha fetoprotein (AFP), albumin, cytokeratin 8 (CK8), and negative for hepatitis B surface antigen (HBsAg) by immunocytochemistry. Fluorescence polymerase chain reaction (PCR) showed HBV DNA integration in the cellular genome. When 5 x 10(6) cells were injected subcutaneously into nude mice, tumorigenicity was 100%, with a latency period of 11+/-1 days. Five weeks after s.c. injection, the pulmonary metastatic rate was 100%, the median number of lung metastases being 121 per mouse. After orthotopic implantation of tumor tissue into nude mouse liver for 35 days, widespread loco-regional and distant metastases occurred, with 100% abdominal wall metastases, 80% intra-abdominal cavity metastases, 100% intrahepatic metastases, 70% diaphragm metastases, and 100% pulmonary metastases. The median number of lung metastatic lesions was 268 per mouse. Gene expression profile of HCCLM3 was compared by cDNA microarray with MHCC97-L, a clonal cell strain from the same parental cell line but with low metastatic potential; 25 differentially expressed genes were identified, 18 of which showed decreased expression and seven increased expression in HCCLM3, including the decreased expression of cell cycle control gene Rb2, mismatch repair gene hMSH2, and signal transduction gene protein kinase C beta2, and increased expression of signal transduction gene MAP kinase, kinase 6. CONCLUSIONS: A new HCC cell line characterized by high pulmonary metastases via s.c. and orthotopic inoculation was established, which provides a new model for the study of liver cancer metastasis. Its gene expression profile could help in the understanding of the mechanism of metastasis and provide potential targets for anti-metastasis intervention.     

Adipocytes differentiation of NIH3T3 cells induced by peroxisome proliferator activation receptor gamma 2 expression]

OBJECTIVE: To express the mouse peroxisome proliferator activated receptor gamma 2(mPPAR gamma 2) in NIH3T3 cells mediated by the recombinant retrovirus and study its function. METHODS: mPPAR gamma 2 gene digested from the recombinant plasmid pcDNA3/mPPAR gamma 2 and confirmed to contain the target gene segment with fluorescence-sequencing was subcloned into retrovirus vector pGCEN to generate the recombinant retrovirus pGCEN/mPPAR gamma 2. The recombinant retrovirus pGCEN/mPPAR gamma 2 and pGCEN were packaged with PA317 cells and anti-G418 clones of PA317 cells were selected. Viral supernatants were collected and used to infect NIH3T3 cells. Peroxisome proliferator activated receptor gamma 2 (PPAR gamma 2)-expressing NIH3T3 cells cultured in the differentiation media containing PPAR gamma activator ETYA were induced into adipocytes. RESULTS: The recombinant retrovirus pGCEN/mPPAR gamma 2 was constructed, 5 x 10(4) CFU/ml of the viral supernatants containing pGCEN/mPPAR gamma 2 and 6 x 10(5) CFU/ml of the viral supernatants containing pGCEN were obtained. mPPAR gamma 2 was expressed in NIH3T3 cells mediated by the recombinant retrovirus. Lipid accumulation obviously existed in PPAR gamma 2-expressing NIH3T3 cells at 10 days postdifferentiation and the lipid-containing cells morphologically resembled the mature adipocytes in vivo. CONCLUSION: An adipocyte differentiation model in vitro was established. The work is the basis for further researches on the molecular mechanism of adipocyte differentiation induced by PPAR gamma 2.     

Histomorphology and ultrastructure of spontaneous pulmonary neoplasms in strain A mice.

The microscopic and ultrastructural characteristics of spontaneous pulmonary neoplasms in strain A (strA) mice are described. Fifty-one spontaneous lung tumors were identified in 34 out of 57, 11-23-month-old male strA/Hen mice. Grossly, all tumors appeared as yellow-white, discrete nodules ranging in size from 1.0-10 mm. Tumor types were randomly distributed throughout the lung; however, the right lung lobes were most frequently involved. Histologically, tumors were classified as adenoma (34/51) or carcinoma (17/51) as defined by standard histopathologic criteria. Adenomas were usually less than 4 mm in diameter and had solid (16/34), papillary (10/34), or mixed (8/34) histologic growth patterns. Carcinomas were usually greater than 4 mm in diameter and had papillary (13/17) or mixed (4/17) histologic growth patterns. Ultrastructurally, benign tumors consisted of solid or papillary areas of neoplastic type II-like cells. Cells comprising malignant tumors had varying ultrastructural characteristics ranging from well-differentiated alveolar cell types to undifferentiated cells having intracytoplasmic osmiophilic dense bodies, vacuoles, or few specialized organelles commonly observed in mature nonneoplastic pulmonary epithelial cells.     

Anti-tumor effects of adenovirus containing human growth hormone sequences in a mouse model of human ovarian cancer

Women with ovarian cancer have a low survival rate and develop resistance to chemotherapy, so new approaches to treatment are needed. We unexpectedly found administration of a replication-deficient adenovirus containing human growth hormone sequences (AdXGH) was beneficial in a mouse model of human ovarian cancer. Intraperitoneal injections of AdXGH prolonged median survival from a mean of 31?±?1.2 to 40?±?1.4?days in immunodeficient SCID mice given SKOV3.ip1 human ovarian cancer cells in the peritoneal cavity. Adenovirus containing human prolactin or del32-71growth hormone sequences had no effect. Repeated injection of growth hormone or implantation of tablets with sustained growth hormone release did not increase survival. Control mice had overlapping tumors throughout the peritoneal cavity and liver and frequent lung metastases 24?days after tumor cell injection. Mice that received two injections of AdXGH had no lung metastases. Mice that received four injections had no lung or liver metastases and peritoneal fibrosis. They did not survive longer than mice that received two injections, but they had enlarged livers with hepatocellular changes, indicating that a limitation of increasing the dose is liver toxicity.     

Comparison of interleukin-12 with lung cancer and malignant lymphoma undergoing autologous peripheral blood stem cell transplantation

PURPOSE: High-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) has become an important treatment for solid tumors including lung cancer. METHODS: We measured IL-12 levels in patients with lung cancer undergoing autologous PBSCT in order to elucidate the role of IL-12 in immune response recovery following stem cell transplantation. RESULTS: Compared to IL-12 levels at 1 week after PBSCT for lung cancer patients, those at 3 weeks were significantly increased ( P<0.01). In contrast, serum IL-12 levels in malignant lymphoma patients did not change significantly. There were no significant differences in levels of other cytokines between 1 week and 3 weeks after transplantation in patients with lung cancer. The frequency of helper/inducer T cells was increased in peripheral blood 1 week after transplantation in both lung cancer and malignant lymphoma patients. There was a significant increase in activated T cell numbers following PBSCT. Furthermore, high levels of other activated T cells persisted in the post-PBSCT period in patients with lung cancer and the number of cytotoxic T cells significantly increased. Natural killer (NK) cell numbers also tended to increase, although that of malignant lymphoma was not significant. A strong correlation was observed between serum IL-12 levels and NK cell numbers and interferon-gamma levels in lung cancer not but in malignant lymphoma patients. The analysis of transfused PBSC showed that the numbers of granulocyte/macrophage colony-forming units were similar in lung cancer and malignant lymphoma patients. However, the number of CD34+ cells was significantly higher in lung cancer than in malignant lymphoma patients. All of the CD34+ subpopulations were lower in percentage in patients with lung cancer than in patients with malignant lymphoma. In particular, the CD34+ CD33- subpopulation was significantly lower in percentage in lung cancer patients. CONCLUSION: Our findings suggest that PBSC in lung cancer are potent mediators of anticancer activity and that they might play an immunotherapeutic role against autologous malignant cells.