Metastatic Clones Research Articles

Circadian coordination of cancer growth and metastatic spread.

To understand whether mammalian circadian time structure measurably affects the host-cancer balance, we studied tumor-take frequency after s.c. tumor cell inoculation and the number of pulmonary tumor nodules after i.v. tumor cell injections at each of 6 equispaced times of day. We employed 2 genetically distinct mouse strains and 2 different tumor model systems, a methylcholanthrene A-induced fibrosarcoma of C(3)HeJ mice and 2 B(16) melanoma cell lines of vastly different metastatic efficiency in C57 Black/6 mice. Fibrosarcoma cells were injected s.c. in 1 of 8 different doses, at 1 of 8 permutated anatomic sites and at 1 of 6 equispaced circadian times, in 96 female C(3)HeJ mice maintained under a synchronizing schedule of 12 hr light alternating with 12 hr dark. Regardless of tumor cell dose and inoculum location, tumor-take frequency depended strongly upon the circadian stage of tumor cell inoculation. Injections of between 2,000 and 50,000 live tumor cells inoculated near the daily sleep/wake interface resulted in the lowest incidence of tumor take compared with inoculation at other times of day. In the experimental i.v. B(16) melanoma metastatic model (N = 110), the capacity of both high and low metastatic potential clones to successfully metastasize to lung depended, to a large extent, upon when in the day each of these clones was injected. Similar to the fibrosarcoma data, the daily sleep/wake boundary was the time of day associated with the greatest resistance to metastatic spread.

Circadian coordination of cancer growth and metastatic spread

To understand whether mammalian circadian time structure measurably affects the host-cancer balance, we studied tumor-take frequency after s.c. tumor cell inoculation and the number of pulmonary tumor nodules after i.v. tumor cell injections at each of 6 equispaced times of day. We employed 2 genetically distinct mouse strains and 2 different tumor model systems, a methylcholanthrene A-induced fibrosarcoma of C3HeJ mice and 2 B16 melanoma cell lines of vastly different metastatic efficiency in C57 Black/6 mice. Fibrosarcoma cells were injected s.c. in 1 of 8 different doses, at 1 of 8 permutated anatomic sites and at 1 of 6 equispaced circadian times, in 96 female C3HeJ mice maintained under a synchronizing schedule of 12 hr light alternating with 12 hr dark. Regardless of tumor cell dose and inoculum location, tumor-take frequency depended strongly upon the circadian stage of tumor cell inoculation. Injections of between 2,000 and 50,000 live tumor cells inoculated near the daily sleep/wake interface resulted in the lowest incidence of tumor take compared with inoculation at other times of day. In the experimental i.v. B16 melanoma metastatic model (N = 110), the capacity of both high and low metastatic potential clones to successfully metastasize to lung depended, to a large extent, upon when in the day each of these clones was injected. Similar to the fibrosarcoma data, the daily sleep/wake boundary was the time of day associated with the greatest resistance to metastatic spread. Int. J. Cancer 83: 365–373, 1999.

SCID-hu mice for the study of human cancer metastasis.

Cancer metastasis involves dynamic and multistep in vivo processes. While generation of metastatic clones requires genetic alterations in cancer cells, subsequent selection of the clones is heavily influenced by interactions with the surrounding tissue microenvironment. To reproduce the complex cellular interactions that occur in human patients is, however, difficult, and has not been achieved using currently available in vitro systems or conventional animal models. The SCID-hu mouse is generated by surgical implantation of human fetal tissues into mutant mice of the severe combined immunodeficient (SCID) phenotype. The unique feature of this model is that the implanted human tissues maintain their normal architecture and function. Therefore implanted human tissues will provide relevant microenvironments for the growth and metastasis of human cancer cells. The SCID-hu mouse model, which was specifically designed for the study of human cancer biology, enables experimental investigation of cellular events involved in cancer metastasis on the basis of interactions between human cancer cells and the human tissue microenvironment. It has been demonstrated that various types of human cancer cell lines generate tumors in implanted human bone marrow and lung, organs frequently involved in metastasis in patients, upon intravenous inoculation. Tumorigenic activity in SCID-hu mice faithfully reflects the clinical features of the original cancer. Tumor formation and selection of high tumorigenic variants occur in a species-specific manner. Furthermore, it was shown that metastatic tumor formation is regulated by both cancer cells and conditions in the host organs. Conditioning of animals by either whole-body irradiation or interleukin 1alpha treatment prior to cancer cell inoculation induced metastatic tumor formation by certain small cell lung cancer (SCLC) cell lines specifically in human bone marrow. A novel gene has been identified by comparing gene expression profiles between high and low tumorigenic SCLC cells in human lung. This gene is preferentially expressed in low metastatic lines, and transfection of the gene into highly metastatic cells results in suppression of metastasis. Recent studies have shown that the gene product is involved in the apoptosis induction pathway. Collectively, our results indicate that the SCID-hu mouse will serve as a unique platform technology with which to investigate cellular events involved in human cancer metastasis, as well as to identify genes playing important roles in the growth and metastasis of human cancer, in the context of interactions between human cancer cells and human tissue environments.

Effects of Tumor Cell-Derived Interleukin 1 Alpha on Invasiveness of Metastatic Clones of Murine RCT Sarcoma through Endothelial Cells

Interleukin 1 alpha (IL-1α) production and invasiveness through mouse lung endothelial cells (MLE) were investigated in high-metastatic RCT+ and low-metastatic RCT– clones established from poorly differentiated murine sarcoma. Apparently, a higher level of IL-1α was derived from RCT+ cells than from RCT– cells. In an invasion assay, the number of cells which penetrated the MLE monolayer in RCT+ was significantly greater than that in RCT–. The invasiveness of RCT+ and RCT– cells was stimulated by additional recombinant mouse IL-1α (rIL-1α) in a dose-dependent manner. Anti-mouse IL-1α monoclonal antibody (anti-IL-1α mAb) significantly inhibited the invasiveness of RCT+ and RCT– cells through the MLE monolayer. However, in RCT+ cells these effects were higher than in RCT– cells. In an attachment assay, the ability of RCT+ cells to attach to the MLE monolayer was significantly higher than that of RCT– cells. The attachment ability of RCT+ and RCT– cells to the MLE monolayer was significantly increased by the pretreatment with rIL-1α in a dose-dependent manner. In a retraction assay, conditioned medium of RCT+ stimulated the retraction of the MLE monolayer more markedly in comparison with conditioned medium of RCT–. The retraction of the MLE monolayer was stimulated by additional rIL-1α in a dose-dependent manner. The increased retraction of the MLE monolayer was closely associated with the enhancement in tumor cell invasiveness. These findings suggest that IL-1α derived from RCT+ and RCT– cells might contribute to the enhancement of tumor cell invasion by stimulating the attachment to the MLE monolayer and retraction of the MLE monolayer.

Metastatic and non-metastatic colorectal cancer (CRC) cells induce host metalloproteinase production in vivo.

Numerous studies have demonstrated the persistent localization of matrix metalloproteinase (MMP) expression to the interface between invading human colorectal cancer (CRC) cells and surrounding stroma supporting a role for MMPs in CRC invasion and metastasis. The present study sought to determine whether CRC cells of varying metastatic potential would have differential effects on host MMP release. Subcutaneous CRC tumors were generated in BALB/c nude mice using three CRC cell lines: SW480, SW620, and the highly metastatic SW620S5 clone. Representative samples from the subcutaneous CRC were then orthotopically implanted on the cecum of recipient nude mice. Subcutaneous and cecal tumors were analyzed for MMP expression via zymography, western blot, and RT-PCR. In vitro, none of the three cell lines expressed MMP-2 nor MMP-9. In contradistinction, the subcutaneous tumors expressed limited amounts of MMP-2 and MMP-9 while the cecal tumors expressed significant amounts of MMP-2 and MMP-9 as well as other smaller members of the MMP family. MMP-9 mRNA and protein was confirmed as host in origin by RT-PCR with mouse specific primers and a mouse MMP-9 molecular weight of 105 kDa as determined by zymography and western blot analysis. In situ hybridization also localized the mRNA for MMP-9 to the host stromal cells. In conclusion, CRC cells appear incapable of producing MMP-2 and MMP-9 in vitro but are capable of up-regulating host MMP production in vivo. Enhanced host MMP-9 production in metastatic CRC cell-derived subcutaneous and cecal tumors suggests that metastatic colon cells may acquire the expression of important MMP regulating factor(s) in vivo.

Role of autocrine motility factor in a 12-lipoxygenase dependent anti-apoptotic pathway.

Apoptosis or programmed cell death, plays an indispensable role in embryonic development, maturation of the immune system and maintenance of tissue and organ homeostasis (1–2). A wide spectrum of molecular entities including oncogenes, tumor suppressor genes, signal transducers, cell cycle proteins, free radicals, cations and proteases have been implicated in apoptosis. Recent work from our laboratory (3) demonstrated that 12-lipoxygenase (12-LOX) may play an essential role in regulating apoptosis in rat Walker carcinosarcoma (W256) cells. Down regulation of 12-LOX gene expression by antisense oligonucleotides triggered time and dose dependent apoptosis. The W256 cell death, triggered by antisense oligos could be partially blocked by exogenous 12(S)-HETE. The antisense oligo treatment also resulted in a significant decrease in the ratio of Bcl-2/Bax proteins which are critical determinants of apoptosis. As expected, over expression of Bcl-2 protein provided partial death sparing effects (3). It also was demonstrated that exogenous arachidonic acid (AA) protected against apoptosis (4). Exogenous AA in a time and dose dependent fashion suppressed NDGA induced W256 apoptosis as well as DNA fragmentation. Serum withdrawal also caused W256 cells to undergo typical apoptosis which was not rescued by several growth factors commonly found in serum. However, exogenous AA suppressed serum starvation induced W256 cell apoptosis (4). Further studies demonstrated that the factor responsible for AA induced protection against apoptosis was the 12-LOX metabolite of AA 12(S)-HETE. 12-LOX has been demonstrated by our laboratory to be expressed in numerous tumor cells from human, rat and mouse (5). Factors controlling the regulation of 12-LOX are largely unknown. Several growth factors have been shown to stimulate 12-LOX activity in tumor cells. For example, in A431, human epidermoid carcinoma cells platelet type 12-LOX is expressed (6). Serum starvation induces a time and dose dependent reduction in the expression of 12-LOX protein (8). Another cytokine which has been demonstrated to regulate 12-LOX levels in tumor cells is autocrine motility factor (AMF). AMF stimulates cell motility and invasion via receptor mediated signaling pathways. AMF is also a potent growth factor. Our laboratory demonstrated that AMF induced cell motility was mediated by the lipoxygenase generation of 12(S)-HETE (7). In a subsequent report, we also demonstrated that AMF was able to increase the cellular protein levels of 12-LOX in high but not in low metastatic clones (8).

Enhanced inhibition of experimental metastasis by the combination chemotherapy of Cu-Zn SOD and adriamycin.

We previously reported that reactive oxygen species (ROS) enhance tumor cell metastasis, and by administration of recombinant human superoxide dismutase (rh SOD), an enzyme which scavenges O2- successfully reduced lung metastasis of mouse MethA sarcoma and Lewis lung carcinoma. These observations suggested that rh SOD suppressed tumor cell invasion by eliminating O2- the primary source of ROS. However, for the clinical application of the drug as an anti metastatic agent, rh SOD needs to be administered in combination with other cytotoxic agents, since SOD by itself has no cytotoxic activity. In this paper, we investigated the effectiveness of the combination chemotherapy of rh SOD and adriamycin (ADR), an anti-cancer agent against the experimental metastasis of highly metastatic clone, MH-02, which was derived from murine Meth A sarcoma. The present metastasis experiment clearly indicates that the administration of rh SOD enhances the antimetastatic effect of ADR. On the other hand, we found that the inhibition rate of metastasis exhibited by the combination chemotherapy of rh SOD and a certain dose (5 mg/ml) of ADR was inferior to that of rh SOD. This apparent paradoxical phenomenon was presumably explained by our finding that tumor cells themselves augment their invasive capacity and platelet aggregation, both of which are causative factors for metastasis formation, by generation of O2- when they were treated with ADR. Nevertheless, the combination chemotherapy of SOD with anticancer drugs such as ADR can be a practical anti-metastasis strategy.

Overexpression of p185erbB2/neu in the NbE prostatic epithelial cell line increases cellular spreading and the expression of integrin alpha6beta1.

Overexpression of p185erbB2/neu has been demonstrated in approximately 30% of prostatic carcinomas and has been shown to induce tumorigenesis and metastasis in a prostatic epithelial cell line, NbE. To metastasize successfully, cells must be able to proliferate, degrade and be motile on a variety of substrates; thus attachment and spreading on a variety of substrates are key features of the metastatic phenotype. Using in vitro assays, we demonstrate that NbE-neuT-9/10 clones attached significantly better to specific substrates and spread significantly better on both specific and non-specific substrates as compared to the control clones. Additionally, the expression of integrin alpha6beta1, a key receptor enabling attachment and spreading on laminin, is upregulated on the metastatic clones NbE-neuT-9 and 10.

Involvement of Liver-Associated Immunity in Hepatic Metastasis Formation

Aims.Hepatic metastasis formation and prevention were studied from the viewpoint of liver-associated immunity.Methods.RCN-9, a colonic cancer cell line derived from Fischer rats, and its subclone RCN-H4, in which the cancer is highly metastatic to the liver, were used. Fischer rats that were inoculated with parent RCN-9 colonic cancer cells (5 × 106) via the portal vein showed liver metastasis in less than 60% of the animals. In contrast, all rats (100%) that received RCN-H4 produced multiple liver metastases. To investigate the difference of hepatic metastasis formation, we assessed the susceptibility of both cell lines against hepatic sinusoidal lymphocytes (HSL) by51Cr-release assay, and the expression of MHC class I and class II of both cell lines by flow cytometry. In addition, we examined whether activation of HSL by interleukin-12 (IL-12) can prevent liver metastasis of highly metastatic clone RCN-H4.Results.The RCN-H4 clone showed decreased susceptibility to lysis by natural cytotoxic cells in HSL. This decrease in susceptibility was attributable to an increase in cell surface expression of MHC class I antigen. Administration of IL-12, a potent NK/CTL stimulatory cytokine, augmented the cytotoxic activity against the RCN-H4 clone and prevented liver metastasis of RCN-H4 inoculated into the portal vein.Conclusions.Liver metastasis formation is positively correlated with the strength of the hepatic immune system which mainly consists of ontogenetically primitive T cells. As these effectors exert their cytotoxicity in a MHC-nonrestricted fashion, tumor cells that highly express MHC class I antigen can readily avoid hepatic surveillance and apt to cause liver metastasis. Augmentation of the hepatic immune system, for instance, with IL-12 administration, can prevent liver metastasis even in tumor cells with a high potential for liver metastasis.

Therapy of cancer metastasis by activation of the inducible nitric oxide synthase.

The process of cancer metastasis consists of multiple sequential and highly selective steps. The vast majority of tumor cells that enter the circulation die rapidly; only a few survive to produce metastases. This survival is not random. Metastases are clonal in origin and are produced by specialized subpopulations of cells that preexist in a heterogeneous primary tumor. Experimental studies concluded that metastatic cells survive in the circulation whereas nonmetastatic cells do not. In part, this difference is due to an inverse correlation between expression of endogenous inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) and metastatic potential. Direct evidence for the role of iNOS in metastasis has been provided by our data on transfection of highly metastatic murine K-1735 clone 4 (C4.P) cells which express low levels of iNOS, with a functional iNOS (C4.L8), inactive mutated iNOS (C4.S2), or neomycin resistance (C4.Neo) genes in medium containing 3 mM of the specific iNOS inhibitor NG-L-methyl arginine (NMA). C4.P, C4.Neo, and C4.S2 cells were highly metastatic, whereas C4.L8 cells were not. Moreover, C4.L8 cells produced slow-growing subcutaneous tumors in nude mice, whereas the other 3 cell lines produced fast-growing tumors. In vitro studies indicated that the expression of iNOS in C4.L8.5 cells was associated with either cytostasis or cytolysis via apoptosis, depending upon NO output. The tumor cells producing high levels of NO underwent autocytolysis and produced cytolysis of bystander cells under both in vitro and in vivo conditions. Multiple i.v. injections of liposomes containing a synthetic lipopeptide upregulated iNOS expression in murine M5076 reticulum sarcoma cells growing as hepatic metastases. The induction of iNOS was associated with the complete regression of the lesions. Transfection of interferon-beta suppressed tumor formation and eradicated metastases, which was apparently linked to iNOS expression and NO production in host cells such as macrophage. Besides mediating cell death, NO produced tumor suppression by regulating expression of genes related to metastasis, e.g., survival, invasion, and angiogenesis. Suppression of metastasis can be achieved through use of immunomodulators that induce iNOS expression in tumor lesions or by the direct delivery of the iNOS gene to tumor cells or host cells through liposome and/or viral vectors.

KAI1, a putative marker for metastatic potential in human breast cancer

The KAI1 gene maps to chromosome 11p11.2, is a metastasis-suppressor gene for human prostate cancer and also is involved in the progression of human pancreatic and non-small cell lung cancer. Recently, we showed that introduction of a single copy of normal, neomycin-tagged human chromosome 11 into highly metastatic MDA-MB-435 breast cancer cells suppressed breast cancer metastasis. Concomitantly, KAI1 levels were higher in chromosome 11/MDA-MB-435 cell clones. The purpose of this study was to test whether KAI1 expression is indicative of breast cancer metastasis using a panel of immortalized breast epithelial and breast cancer cell lines that represent multiple stages of breast cancer progression. Metastatic cell clones isolated from the parental mixed, wild-type population of MDA-MB-435 cells expressed the lowest levels of KAI1 mRNA and chromosome 11 containing MDA-MB-435 (neol1/MDA-MB-435.A3 and neol1/MDA-MB-435.B1) cells had approximately twice as much KAI1 mRNA than the parental clones. MCF-10A, an immortalized normal-like non-tumorigenic mammary epithelial cell line, had the highest level of KAI1 mRNA. We compared the metastatic propensity and invasive ability of a continuum of breast cancer cells with varying degrees of progression toward malignancy and found that these parameters tended to correlate inversely with KAI1 mRNA expression. These data suggest that, in addition to its role in human prostate, pancreatic and non-small cell lung cancer, KAI1 may also be a useful marker for staging human breast disease.

Modification of PDGFalpha receptor expression or function alters the metastatic phenotype of 3LL cells.

Functional PDGFalpha receptors are selectively expressed on highly lung-metastasizing clones of the 3LL Lewis lung carcinoma, but not on low-mestastatic clones. The highly metastatic clones are also growth induced in vitro by PDGF and lung conditioned medium. To investigate whether modification of PDGFalpha receptor expression or function can affect metastatic capability, we transfected cells of a low-metastatic 3LL clone with a full length PDGFalpha receptor gene and cells of a highly-metastatic clone with a truncated kinase domain PDGFalpha receptor gene. Introduction of the full length PDGFalpha receptor conferred upon low-metastatic cells the ability to grow in vitro in the presence of PDGF-AA and to colonize the lung in experimental and spontaneous metastases assays. Conversely, introduction of a truncated version of the PDGFalpha receptor into highly metastatic cells reduced their metastatic load to control levels. Accordingly, their responses to PDGF-AA, including growth stimulation and receptor autophosphorylation, were reduced. These results demonstrate that PDGFalpha receptor expression and function can control the capacity of tumor cells to generate metastases in the lung. The response of this receptor to lung-derived PDGF-like factors may define a paracrine mode of metastatic cell growth in the target organ.

Cytoplasmic microtubules in two different mouse melanoma cell lines: a qualitative and quantitative analysis using confocal laser scanning microscopy and computer-assisted image analysis.

The microtubular system as one part of the cellular cytoskeleton is not only necessary for mitotic activity of malignant cells but also for invading neighboring tissues and for the formation of distant metastases. In the present study, the amount and distribution of tubulin in two murine melanoma cell lines (K1735-M2: high metastatic clone; K1735-c116: low metastatic clone) were determined quantitatively using an indirect immunofluorescence technique, confocal laser scanning microscopy (CLSM) and computer-assisted image analysis. Additionally, qualitative and quantitative changes after application of the microtubule-inhibitor nocodazole were investigated. Quantitative analysis showed a significant difference between the high and low metastatic cell line for the parameter TEXTURE, indicating a finer structured network within the high metastatic cells. After treatment with nocodazole the parameters TEXTURE and DENSITY were reduced, suggesting a decrease of assembled tubulin and a less delicate structure of the remaining microtubules. Our study shows that CLSM combined with computer-assisted image analysis provides a new method to examine quantitative variations of the cytoskeleton possibly related to cell function.

Resistance to IL-1 anti-proliferative effect, accompanied by characteristics of advanced melanoma, permits invasion of human melanoma cells in vitro, but not metastasis in the nude mouse.

We reported earlier that IL-1 inhibits the growth of human melanoma cells (A375-6), and that these cells become resistant to IL-1 after prolonged periods of culture. The resistant cells constitutively produce IL-alpha and IL-6 with IL-6 production was induced by endogenous IL-1 in an autocrine manner. The cells are also resistant to IL-6 anti-proliferative effects. In the present study, we show that the resistant clones exhibited up-regulated expression of intercellular-adhesion molecule 1 (ICAM-1) and vitronectin receptor (integrin alpha(v)beta3) when compared with the IL-1-sensitive clone, A375-6. Moreover, these IL-1-resistant clones exhibited many other metastatic characteristics, such as expression of IL-8 mRNA, production of matrix metalloproteinases (MMP-2 and MMP-9), and augmented invasion activity. However, contrary to our expectations, the IL-1-resistant cells did not exhibit experimental metastasis in a nude-mouse model, similarly to the IL-1-sensitive parental A375-6 cell line. In contrast, the highly metastatic clone A375-SM exhibited alpha(v)beta3 expression at a level comparable to that of the IL-1-resistant cells, but expressed low or no ICAM-1, metalloproteinase and displayed little in vitro invasion activity. These results show that the metastatic characteristics of IL-1-resistant cells are not sufficient to produce metastasis in vivo and suggest that these resistant clones may provide a good model system for characterizing the molecular mechanisms of metastasis.

Metastatic ability of MXT mouse mammary subpopulations correlates with clonal expression and/or membrane-association of gelatinase A.

We have developed a novel murine mammary tumor system with variants representing different stages of tumor progression. The MXT-s parental cell line was established from a urethane-induced and hormone-sensitive mammary tumor. MXT-s parental cells are highly tumorigenic but poorly metastatic. MXT clones and variants were selected by either in vitro or in vivo procedures, and they differ in metastatic ability and 17 beta-estradiol dependency for tumor growth. The MXT-c1.1 and MXT-B2 cell lines produced lung metastasis after intravenous injection into 100% of syngenic mice, but only MXT-c1.1 cells were highly metastatic from intramammary tumors. The fingerprints obtained by arbitrarily primed-polymerase chain reaction demonstrated that the metastatic variants and clones had a common genetic background and resulted from clonal selection from the parental cell line. We studied whether the matrix metalloproteinase (MMP) profile is correlated with tumor progression and metastatic ability in the MXT tumor system. Gelatinases A and B were assayed in the cells, both by enzyme activity and mRNA expression. Gelatinase A was expressed in MXT-c1.1 cells, whereas MXT-B2 cells did not express either MMP. In contrast, the mammary fat pad tumors expressed both gelatinases. Membrane Type 1-MMP transcripts were also detected in MXT cells and tumors. Because the mRNA levels of gelatinase. A were low in MXT-B2 tumors, we suggested that exogenous gelatinase A bound the cell membranes of MXT-B2 cells in vivo. Indirect evidence was obtained in vitro by treatment of MXT-B2 cells with NIH/3T3 fibroblast-conditioned medium. After this treatment, we detected a gelatinolytic activity at M(r) 68,000 in the cell-membrane extract of MXT-B2 cells and an increase in migratory ability through type IV collagen matrices. On the other hand, Ha-ras gene dosage correlated positively with metastatic ability but not with either gelatinase A or gelatinase B expression. No significant differences were observed in the expression of stromelysin-1 and tissue inhibitors of MMP. Thus, in the MXT tumor system, the expression of gelatinase A or its cell association and Ha-ras gene dosage independently contribute to the metastatic phenotype.

Direct Correlation Between DNA Repair Capacity and Metastatic Potential of K-1735 Murine Melanoma Cells

The purpose of this study was to determine whether the ability of K-1735 murine melanoma cells to repair DNA damage correlates with their metastatic potential. Three nonmetastatic clones, four metastatic clones, and three somatic-cell hybrids between metastatic and nonmetastatic clones were exposed to incident ultraviolet (UV) light (254 nm). Cell survival was determined by the microculture tetrazolium assay, which measures cell metabolic activity. DNA repair capacity was determined with a host-cell reactivation assay, which measures the activities of chloramphenicol acetyltransferase encoded by the reporter gene in both UV-damaged and undamaged plasmid (control) pCMV cat 40 h after transfection. No discernible differences in transfection efficiencies were found between K-1735 clones with low and high metastatic potential or between cells transfected with UV-damaged and control plasmids. DNA repair capacity directly correlated with cell survival (p < 0.05) and with metastatic potential in the K-1735 clones and somatic cell hybrids (p < 0.05). These data suggest that the intrinsic resistance of melanoma metastases to systemic chemotherapy may be due, in part, to the cells' enhanced DNA repair capacity.

Expression of basic fibroblast growth factor is necessary but insufficient for production of metastasis

We determined whether overexpression of basic fibroblast growth factor (bFGF) is necessary for enhanced growth and production of metastasis by murine K-1735 melanoma cells. The bFGF gene was transfected into three nonmetastatic clones (C-IO, C-19, and C-23) that do not express bFGF mRNA and protein and one metastatic clone that expresses high levels of bFGF mRNA and protein. Control cells were transfected with a dominant selectable marker neomycin resistance gene (neo). All bFGF-transduced cells expressed bFGF-specific mRNA transcripts and cellular bFGF protein and proliferated in culture with medium containing low concentrations of serum. Anchorage-independent growth in hard agarose was enhanced only in bFGF-transfected nonmetastatic C-10 cells which, subsequent to the transfection, also expressed high levels of collagenase IV/gelatinase A activity. The treatment of C-10, C-19, and C-23 cells with exogenous bFGF induced collagenase IV/gelatinase expression, as did the addition of lysates from C-10/bFGF and C-23/bFGF cells. C-10/bFGF cells (but not C-19/bFGF or C-23/bFGF) produced highly vascular and rapidly growing subcutaneous tumors as well as a high incidence of lung metastasis. These data suggest that overexpression of bFGF is necessary but in itself not sufficient to convert nonmetastatic K-1735 cells to the metastatic phenotype and that enhanced tumorigenicity and metastasis require at least concurrent expression of bFGF and collagenase type TV genes.

Enhanced metastasis of B16 melanoma cells by unexpected elevated expression of the metastasis-associated TI-241 (LRF-1-, Jun-Fos-related) gene treated with antisense oligonucleotide.

B16-F10 is a B16 mouse melanoma subline that preferentially metastasizes to the lung following intravenous injection. Previously we isolated TI-241 (LRF-1 homologue related to Jun-Fos) gene that was expressed higher in the high metastatic clone B16-F10 than the low metastatic clone F1. Transfection of TI-241 into F1 converted it into a high-metastatic cell. We studied the effect of antisense oligonucleotide designed to reduce the expression of TI-241 in B16-F10 cells, and observed an unexpected increase in the TI-241 level. The increase in the expression was maximal at 30 h, then it decreased during further culture with or without TI-241 antisense oligonucleotide. This increased TI-241 expression by antisense oligonucleotide was also observed in B16-F1 cells whereas sense oligonucleotide did not affect the expression. B16-F10 cells cultured with TI-241 antisense oligonucleotide showed enhanced experimental metastatic potential to the mouse lungs compared with untreated B16-F10 and B16-F10 cultured with TI-241 sense oligonucleotide.

Enhancement of lung-colonizing potential of murine tumor cell lines co-cultivated with activated macrophages.

In order to explore the influence of activated macrophages on tumor cells, we cultured a series of weakly metastatic clones isolated from the murine T3 fibrosarcoma line (T3 clones) and the B16-F10 melanoma cells on feeder layers of C. parvum- or thioglycollate-elicited macrophages, or 'resident' (unstimulated) macrophages. Co-cultivation with C. parvum-elicited macrophages, but not with resident macrophages, induced an increase of the lung-colonizing potential of T3 clones and B16-F10 cells. An enhancement of lung-colonizing potential was also found in tumor cells grown in media conditioned by C. parvum-elicited macrophages. Thioglycollate-elicited macrophages also generated a pro-clonogenic activity which was however effective only on T3 clones but not on B16-F10 cells. The increase in the lung-colonizing potential of tumor cells stimulated by activated macrophages was retained to some degree after subcultivation in tissue culture medium or transplantation into syngeneic animals. In conclusion, our data support the notion that macrophages at different states of activation may enhance lung colonization of tumor cells by inducing a partially stable change of phenotype.

Alterative expression of the collagenase and adhesion molecules in the highly metastatic clones of human colonic cancer cell lines.

Human colonic carcinoma cell lines, KM12C, KM12SM and KM12L4, were previously established and their in vivo metastatic potentials have been well evaluated. The highly metastatic cell lines KM12SM and KM12L4 were derived from the parental low metastatic cell line KM12C in vivo. To evaluate the metastatic behavior of these cell lines in vitro, we examined colony formation on monolayers of the pulmonary arterial endothelial (CPAE) cells. On day 4, the highly metastatic cell lines showed an approximately 2-fold increase in number of colonies on CPAE cell monolayers relative to the parental KM12C cell line. To investigate what evidence is correlated with their metastatic and invasive abilities, Northern blot analysis and flow cytometry were performed in all cell lines. According to the results of Northern blot analysis, the levels of matrix metalloproteinase (MMP)-2 and c-met mRNA expression were increased in highly metastatic cell lines as compared with the parental cell line. We also examined the cell-surface expression of several adhesion molecules by flow cytometry. The levels of expression of sialyl Lewisa antigen (sLe(a)) in KM12SM and KM12L4 were twice higher than that in KM12C. However, the levels of expression of E-cadherin in KM12SM and KM12L4 were decreased to half that in KM12C. The alterative expression of the collagenase and adhesion molecules might contribute to their metastatic/invasive abilities of these cell lines both in vivo and in vitro.