BMP2 signalling activation enhances bone metastases of non‐small cell lung cancer

Distant metastases occur when non‐small cell lung cancer (NSCLC) is at late stages. Bone metastasis is one of the most frequent metastases of NSCLC and leads to poor prognosis. It has been reported that high expression of BMP2 in NSCLC correlates with poor survival, but whether BMP2 contributes to NSCLC bone metastasis remains largely unknown. The activation of BMP signalling is found in metastatic bone tumours of mice Lewis lung carcinoma and predicts poor survival in human NSCLC. BMP2 signalling activation can enhance bone metastasis of Lewis lung carcinoma. Moreover, BMP2 secreted by stroma fibroblasts can promote the migration and invasion of NSCLC cells. Besides, in combination with pre‐osteoblast and LLCs, BMP2 could enhance the differentiation of macrophages into osteoclasts to play roles in the osteolytic mechanism of NSCLC bone metastasis. Interestingly, NSCLC cells can also enrich BMP2 to pre‐osteoblasts to function in the osteoblastic mechanism. Our results firstly demonstrate the detailed mechanisms about what roles BMP2 signalling play in enhancing NSCLC bone metastases. These findings provide a new potential therapy choice for preventing bone metastases of NSCLC via the inhibition of BMP2 signalling.     

Effect of Z-100, an immunomodulator extracted from human type tubercle bacilli,on the pulmonary metastases of lewis lung carcinoma in attempt to regulate suppressor T cells and suppressor factor,IL-4

In the present study, anti-metastatic effect of Z-100 on the spontaneous pulmonary metastases of Lewis lung carcinoma (3LL) was examined in an attempt to regulate suppressor T cells. When Z-100 (10 mg/kg) was daily injected i.p. after 3LL inoculation, survival rate of these mice was increased significantly (p<0.05). In addition, the number of pulmonary metastatic colonies of 3LL in Z-100-treated mice were significantly decreased by 38% at 21 days, as compared with that of control mice (p<0.05). Along with the decrease of pulmonary metastases, suppressor cell activity was also gradually reduced in these mice, as compared with that of control mice. When splenic suppressor cells (5×10⁷ cells) from 3LL-bearing mice were adoptively transferred into normal mice (recipients) just before inoculation of 3LL, the development of pulmonary metastases in recipients was significantly accelerated. However, splenocytes from 3LL-bearing mice treated with Z-100 did not affect the development of pulmonary metastasis. The potential to accelerate the metastasis of splenic mononuclear cells from 3LL-bearing mice was decreased significantly by the treatment with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb followed by complement. IL-4 activity in the sera of 3LL-bearing mice was detected 15 days after tumor inoculation (13 pg/ml) and gradually increased (18 pg/ml) 20 days after tumor inoculation. However, when Z-100 (10 mg/kg) was daily injected i.p., IL-4 activity in sera was decreased significantly, and the IL-4 activity was not detected in these mice on day 20. These results suggest that Z-100 could inhibit the pulmonary metastases in 3LL-bearing mice through the inhibition of suppressor T cell activity and a possible candidate of its effector molecule, IL-4.     

T cell-derived IL-10 promotes lung cancer growth by suppressing both T cell and APC function.

We have found previously that human lung cancers potently induce T lymphocyte IL-10 production in vitro. To assess the impact of enhanced T cell-derived IL-10 on antitumor immunity in vivo, we utilized transgenic mice expressing IL-10 under the control of the IL-2 promoter. We have shown previously that Lewis lung carcinoma cells (3LL) have more aggressive growth potential in IL-10 transgenic mice compared with control littermates. In this study, we show that transfer of T cells from IL-10 transgenic mice to control littermates transferred the IL-10 immunosuppressive effect and led to enhanced 3LL tumor growth. In addition to changes in T cell-mediated immunity, professional APC from IL-10 transgenic mice were found to have significantly suppressed capacity to induce MHC alloreactivity, CTL responses, and IL-12 production. Tumor Ag-pulsed dendritic cells from IL-10 transgenic mice also failed to generate antitumor reactivity. These results suggest that increased levels of T cell-derived IL-10 severely impair antitumor immunity in vivo, due to defects in both T cell and APC function.     

Specific inhibition of cyclooxygenase 2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis

Cyclooxygenase-2 (COX-2), the enzyme at the rate-limiting step of prostanoid production, has been found to be overexpressed in human lung cancer. To evaluate lung tumor COX-2 modulation of antitumor immunity, we studied the antitumor effect of specific genetic or pharmacological inhibition of COX-2 in a murine Lewis lung carcinoma (3LL) model. Inhibition of COX-2 led to marked lymphocytic infiltration of the tumor and reduced tumor growth. Treatment of mice with anti-PGE2 mAb replicated the growth reduction seen in tumor-bearing mice treated with COX-2 inhibitors. COX-2 inhibition was accompanied by a significant decrement in IL-10 and a concomitant restoration of IL-12 production by APCs. Because the COX-2 metabolite PGE2 is a potent inducer of IL-10, it was hypothesized that COX-2 inhibition led to antitumor responses by down-regulating production of this potent immunosuppressive cytokine. In support of this concept, transfer of IL-10 transgenic T lymphocytes that overexpress IL-10 under control of the IL-2 promoter reversed the COX-2 inhibitor-induced antitumor response. We conclude that abrogation of COX-2 expression promotes antitumor reactivity by restoring the balance of IL-10 and IL-12 in vivo.     

Increased local vascular endothelial growth factor expression associated with antitumor activity of proteasome inhibitor

Inhibition of the proteasome, a multicatalytic proteinase complex, is an attractive approach to cancer therapy. Here we report that a selective inhibitor of the chymotrypsin-like activity of the proteasome, PSI (N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal) may inhibit growth of solid tumors not only through apoptosis induction, but also indirectly--through inhibition of angiogenesis. Two murine tumors: colon adenocarcinoma (C-26) and Lewis lung carcinoma (3LL) were chosen to study the antitumor effect of PSI. In an in vivo model of local tumor growth, PSI exerted significant antitumor effects against C-26 colon carcinoma, but not against 3LL lung carcinoma. Retardation of tumor growth was observed in mice treated with both 10 nmoles and 100 nmoles doses of PSI and in the latter group prolongation of the survival time of tumor-bearing mice was observed. PSI inhibited angiogenesis in the C-26 growing tumors with no such effect in 3LL tumors. Unexpectedly, that activity was associated with upregulation of vascular endothelial growth factor (VEGF) at the level of mRNA expression and protein production in C-26 tumors treated with PSI. C-26 cells treated with PSI produced increased amounts of VEGF in vitro in a dose- and time-dependent manner. We demonstrated that in C-26 colon adenocarcionoma higher VEGF production may render endothelial cells susceptible to the proapoptotic activity of PSI and is associated with inhibition of tumor growth.     

Inhibition of HDAC6 Attenuates Tumor Growth of Non-Small Cell Lung Cancer

Histone deacetylase 6 (HDAC6) regulates cytoplasmic signaling networks through deacetylation of various cytoplasmic substrates and serves as a key member of the ubiquitin proteasome system (UPS). This study is focused on HDAC6 regulation of the Notch1 receptor that plays a crucial role in tumor growth in NSCLC. A series of cell culture experiments were employed using A549, Lewis lung carcinoma 2 (LL2), and H1299 NSCLC cell lines to investigate HDAC6-mediated regulation of the Notch1 receptor through the UPS. HDAC6 was inhibited with small molecule inhibitors tubacin and ACY1215 in vitro and in vivo. Inhibition of HDAC6 led to reduced levels of Notch1 receptor in a dose-dependent manner in all three NSCLC cell lines tested. HDAC6 inhibition with ACY1215 led to G2 arrest, increased apoptosis, and increased levels of cleaved PARP1 in A549, LL2, and H1299 cell lines. In vivo inhibition of HDAC6 with ACY1215 significantly reduced LL2 tumor growth rate. Our data show that HDAC6 in NSCLC cells supports Notch1 signaling and promotes cell survival and proliferation. Our results support clinical investigation of HDAC6 inhibitors as a potential therapeutic option for treatment of NSCLC patients.     

Improved survival in tumor-bearing SCID mice treated with interferon-γ-inducible protein 10 (IP-10/CXCL10)

Tumor growth requires angiogenesis, which in turn requires an imbalance in the presence of angiogenic and angiostatic factors. We have shown that the CXC chemokine family, consisting of members that are either angiogenic or angiostatic, is a major determinant of tumor-derived angiogenesis in non-small-cell lung cancer (NSCLC). Intratumor injection of interferon-inducible protein 10 (IP-10, or CXCL10), an angiostatic CXC chemokine, led to reduced tumor growth in a SCID mouse model of NSCLC. In this study, we hypothesized that treatment with CXCL10 would, by restoring the angiostatic balance, improve long-term survival in NSCLC-bearing SCID mice. To test this hypothesis, A549 NSCLC cells were injected in the subcutis of the flank, followed by intratumor injections with CXCL10 continuously (group I), or for ten weeks (group II), or a control group (human serum albumin). Median survival was 169, 130, and 86 days respectively (P<0.0001). We extended these studies to examine the mechanism of prolonged survival in CXCL10-treated mice. CXCL10 treatment inhibited lung metastases, but was dependent upon continued treatment, and was associated with an increased rate of apoptosis in the primary tumor, with no direct effect on the proliferation of the NSCLC cells. Furthermore, the inhibition of lung metastases was due to the angiostatic effect of CXCL10 on the primary tumor, since the rate of apoptosis within lung metastases was unaffected. These data suggest that anti-angiogenic therapy of human lung cancer should be continued indefinitely to realize persistent benefit, and confirms the anti-metastatic capacity of localized angiostatic therapy.     

Selection of 3LL tumor subline resistant to natural effector cells concomitantly selected for increased metastatic potency

Summary Studies were carried out to test whether the selection of 3LL tumor cells resistant to the cytotoxic activity of normal spleen cells will concomitantly select for increased metastatic capacity. A total of 0.5×10⁶ 3LL tumor cells and 50×10⁶ normal spleen cells were admixed and inoculated SC in C57BL/6 mice. This procedure was repeated for eight transplant generations. The tumor cells which were selected in this manner (3LLN) were relatively resistant in vitro to the cytotoxic effect of normal spleen sells. The 3LLN cells were less susceptible than the original 3LL tumor cells to the hybrid resistance mechanisms, as determined by their growth capacity in F1(BALB/c×C57BL/6) or F1(C3H/DiSn×C57BL/6) mice. After three selection generations, 3LLN tumor cells were more efficient in the production of spontaneous lung metastases. These results support our suggestion that natural cell-mediated immunity may be involved in the control of metastatic spread.     

Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer

Lewis lung carcinoma (3LL) cells were constitutively resistant to Fas-mediated apoptosis, but overexpression of Fas on 3LL cells allowed Fas-mediated apoptosis after crosslinking with agonist anti-Fas antibody (Jo2) in vitro. Surprisingly, Fas-overexpressing 3LL cells showed enhanced in vivo tumor progression, whereas no promotion of in vivo tumor growth was observed for dominant negative (DN) Fas-overexpressing 3LL transfectants in which the cytoplasmic death domain was deleted. In addition, the promotion of in vivo tumor growth by Fas-overexpression was reduced in gld (FasL-mutation) mice compared to normal mice. These data indicate that intact Fas/FasL cell signaling is required for the promotion of in vivo tumor growth by Fas overexpression in 3LL cells. In contrast to the efficient Fas-mediated killing induced in vitro by crosslinking with anti-Fas antibody, Fas-overexpressing 3LL cells were resistant in vitro to Fas-mediated apoptosis by activated T cells or transient FasL transfection. These data suggest that agonist anti-Fas antibody and natural FasL can transmit qualitatively different signals, and crosslinking of Fas with natural FasL on 3LL cells does not deliver the expected death signal. Thus, our results demonstrate that in some cases overexpression of Fas can result in a survival advantage for tumor cells in vivo.     

Tumor-associated antigenic differences between the primary and the descendant metastatic tumor cell populations

The existence of antigenic differences between cell populations in the local growth of the 3LL tumor (L-3LL) and its lung metastases (M-3LL) was studied. Normal C57BL/6 spleen cells sensitized in vitro for 5 days against L-3LL monolayers lysed preferentially L-3LL targets but not M-3LL tumor cell targets. Conversely, anti-M-3LL-sensitized lymphocytes killed M-3LL targets more efficiently than they killed L-3LL targets. Furthermore, spleen cells from mice bearing subcutaneous L-3LL tumors were significantly more cytotoxic to L-3LL targets than to M-3LL targets and vice versa. M-3LL cells were found also to be more resistant in vitro and in vivo to natural killer cells than were L-3LL tumor cells. M-3LL cells were more resistant than L-3LL cells to hybrid resistant mechanisms when they were inoculated into F₁ (C3Heb X C57BL/6) or F₁ (BALB/c X C57BL/6) mice. Anti-M-3LL lymphocytes generated both in vitro and in vivo, but not anti-L-3LL lymphocytes, admixed with L-3LL or M-3LL tumor cells and inoculated into footpads of syngeneic recipients suppressed the development of lung metastases. These results suggest that metastatic cells are indeed phenotypic variants of the local growing tumor cell populations. Presumably, these variants are selected for their capacity to home to and grow in the lungs, and for their resistance to specific immune effects initially evoked against the local tumor and to nonspecific natural killer cells. These data may prove to be of importance with respect to any rational approach to the problem of immunotherapy.     

The effect of combination therapy of radiation and Z-100, an arabinomannan on tumor growth in mice

The effect of radiation combined with intraperitoneal administration of Z-100, an immunomodulatory arabinomannan extracted fromMycobacterium tuberculosis, was studied using Meth A fibrosarcoma in BALB/c mice and metastasis of Lewis lung carcinoma, 3LL, in C57BL/6 mice.In mice bearing Meth A fibrosarcoma, a moderate degree of growth inhibition was observed in the group of single therapy with Z-100 or radiation (10 Gy). When radiation was combined with Z-100, the tumor growth was significantly inhibited. In mice bearing 3LL, slight inhibition of pulmonary metastasis was observed in the group of single therapy, while significant degrees of inhibition of primary tumor growth and pulmonary metastasis were observed in the combination group. This suggests the usefulness of combined use of Z-100 in radiation therapy.Abbreviations 3LL Lewis lung carcinoma - IL-3 Interleukin-3 - BRM(s) Biological response modifier(s) - N-CWS Nocardia-Cell wall skeleton - CSF Colony stimulating factor     

Development of a metastatic fluorescent Lewis Lung carcinoma mouse model: Identification of mRNAs and microRNAs involved in tumor invasion

Cancer metastasis is the foremost cause of death in cancer patients. A series of observable pathological changes takes place during progression and metastasis of cancer, but the underlying genetic changes remain unclear. Therefore, new approaches are required, including insights from cancer mouse models. To examine the mechanisms involved in tumor metastasis, we first generated a stably transfected Lewis Lung carcinoma cell line expressing a far-red fluorescent protein, called Katushka. After in vivo growth in syngeneic mice, two fluorescent Lewis Lung cancer subpopulations were isolated from primary tumors and lung metastases. The metastasis-derived cells exhibited a significant improvement in in vitro invasive activity compared to the primary tumor-derived cells, using a quantitative invasion chamber assay. Moreover, expression levels of 84 tumor metastasis-related mRNAs, 88 cancer-related microRNAs as well as Dicer and Drosha were determined using RT-qPCR. Compared to the primary Lewis Lung carcinoma subculture, the metastasis-derived cells exhibited statistically significantly increased mRNA levels for several matrix metalloproteinases as well as hepatocyte growth factor (HGF) and spleen tyrosine kinase (SYK). A modest decrease in Drosha and Dicer mRNA levels was accompanied by significant downregulation of ten microRNAs, including miR-9 and miR-203, in the lung metastatic Lewis Lung carcinoma cell culture. Thus, a tool for cancer metastasis studies has been established and the model is well suited for the identification of novel microRNAs and mRNAs involved in malignant progression. Our results suggest that increases in metalloproteinase expression and impairment of microRNA processing are involved in the acquirement of metastatic ability.     

Suppression of the metastatic potential of oncogene v-src-transformed cells as a result of activity of the exogenous DAP kinase

Hamster tumor cell lines obtained with the Rous sarcoma virus and characterized by a high metastatic activity in vitro were transfected with the gene for C2+/calmodulin-dependent serine-threonine death-associated protein kinase (DAPk). Expression of DAPk in tumor cells dramatically reduced their survival in the blood of syngenic animals and their ability to produce metastases, but did not affect their tumorigenicity or the primary tumor growth. The DAPk-induced change in the metastatic phenotype was not accompanied by substantial changes in production and phosphorylation of v-Src or focal adhesion proteins (focal adhesion kinase and paxilline). The resulting system of transfected cells with a modulated metastatic potential provide a convenient model to study the molecular mechanisms of tumor progression at various steps.     

Morphological appearance, content of extracellular matrix and vascular density of lung metastases predicts permissiveness to infiltration by adoptively transferred natural killer and T cells

We have recently shown that adoptively transferred, IL-2-activated natural killer (A-NK) cells are able to eliminate well-established B16-F10.P1 melanoma lung metastases. However, some B16-F10.P1 lung metastases were resistant to infiltration by the A-NK cells and also resistant to the A-NK cell treatment. The infiltration-resistant (I-R) B16-F10.P1 metastases had a unique “compact” morphology compared to the “loose” morphology of the infiltration-permissive (I-P) metastases. Here, we show that I-P loose tumors and I-R compact tumors are also found in lung metastases of mouse Lewis lung carcinoma (3LL), MCA-102 sarcoma, and MC38 colon carcinoma as well as rat MADB106 mammary carcinoma origin. Furthermore, the infiltration resistance of the compact tumors is not restricted to A-NK cells, since PHA and IL-2 stimulated CD8+ T-cells (T-LAK cells) also infiltrated the compact tumors poorly. Analyses of tumors for extracellular matrix (ECM) components and PECAM-1⁺ vasculature, revealed that the I-R lesions are hypovascularized and contain very little laminin, collagen and fibronectin. In contrast, the I-P loose tumors are well-vascularized and they contain high amounts of ECM components. Interestingly, the distribution pattern of ECM components in the I-P loose tumors is almost identical to that of the normal lung tissue, indicating that these tumors develop around the alveolar walls which provide the loose tumors with both a supporting tissue and a rich blood supply. In conclusion, tumor infiltration by activated NK and T cells correlates with the presence of ECM components and PECAM-1⁺ vasculature in the malignant tissue. Thus, analysis of the distribution of ECM and vasculature in tumor biopsies may help select patients most likely to benefit from cellular adoptive immunotherapy.     

Alpha IIb beta 3 integrin expression and function in subpopulations of murine tumors.

Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G2/M phase of the cell cycle. Both B16a and 3LL cells express alpha IIb beta 3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of alpha IIb beta 3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with alpha IIb beta 3-specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that alpha IIb beta 3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the alpha IIb beta 3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.     

Suppression of the Metastatic Potential of v-src-Transformed Cells by the Exogenous DAP Kinase Gene

Hamster tumor cell lines obtained with the Rous sarcoma virus and characterized by a high metastatic activity in vitro were transfected with the gene for Ca²⁺/calmodulin-dependent serine–threonine death-associated protein kinase (DAPk). Expression of DAPk in tumor cells dramatically reduced their survival in the blood of syngenic animals and their ability to produce metastases, but did not affect their tumorigenicity or the primary tumor growth. The DAPk-induced change in the metastatic phenotype was not accompanied by substantial changes in production and phosphorylation of v-Src or focal adhesion proteins (focal adhesion kinase and paxilline). The resulting system of transfected cells with a modulated metastatic potential provide a convenient model to study the molecular mechanisms of tumor progression at various steps.     

Suppression of proliferation,tumorigenicity and metastasis of lung cancer cells after their transduction by interferon-beta gene in baculovirus vector

BackgroundGene therapy represents an interesting alternative treatment for cancers. Interferon-beta is well known as a multifunctional cytokine that provides antiviral, antiproliferative, antiangiogenic and immunomodulating effects. For this reason introduction of this cytokine gene in baculovirus vector is seen as a rather promising tool for anticancer therapy.AimInvestigation of biological behavior in vitro and in vivo of lung cancer cells modified by interferon-beta gene which was introduced into the cells in vitro with baculovirus vector.Materials and MethodsStudies were performed on mouse Lewis lung carcinoma cells as the tumor model (LL cell line). Transductions of cells by recombinant baculoviruses, in vitro and in vivo analysis of tumor cell biology and immunocytochemical method have been used.ResultsThe study of various in vitro biological parameters of LL cancer cells transduced by recombinant baculovirus with interferon gene demonstrated that the transduction of cells is accompanied by significant inhibition of their proliferation and ability to form colonies in semisolid agar. Also, transduction of LL cells with interferon gene inhibited their tumorigenicity, i.e. the ability to cause formation of tumors and metastases in lungs of mice in vivo. Anti-tumor activity of recombinant interferon is realized via high level of its local production in tumors, induced by LL carcinoma cells, transduced with recombinant interferon-beta gene. Recombinant baculovirus without interferon gene did not influence significantly on tumorigenicity and metastatic ability of lung cancer cells.ConclusionsIntroduction of interferon-beta gene in Lewis lung carcinoma cells in vitro in recombinant baculovirus leads to inhibition of their proliferation potential and malignant behavior in vitro, tumorigenicity and metastatic activity in vivo.     

Depletion of CXCR2 inhibits tumor growth and angiogenesis in a murine model of lung cancer

The Glu-Leu-Arg(+) (ELR(+)) CXC chemokines are potent promoters of angiogenesis and have been demonstrated to induce a significant portion of nonsmall cell lung cancer-derived angiogenic activity and support tumorigenesis. ELR(+) CXC chemokines share a common chemokine receptor, CXCR2. We hypothesized that CXCR2 mediates the proangiogenic effects of ELR(+) CXC chemokines during tumorigenesis. To test this postulate, we used syngeneic murine Lewis lung cancer (LLC; 3LL, H-2(b)) heterotopic and orthotopic tumor model systems in C57BL/6 mice replete (CXCR2(+/+)) and deficient in CXCR2 (CXCR2(-/-)). We first demonstrated a correlation of the expression of endogenous ELR(+) CXC chemokines with tumor growth and metastatic potential of LLC tumors. Next, we found that LLC primary tumors were significantly reduced in growth in CXCR2(-/-) mice. Moreover, we found a marked reduction in the spontaneous metastases of heterotopic tumors to the lungs of CXCR2(-/-) mice. Morphometric analysis of the primary tumors in CXCR2(-/-) mice demonstrated increased necrosis and reduced vascular density. These findings were further confirmed in CXCR2(+/+) mice using specific neutralizing Abs to CXCR2. The results of these studies support the notion that CXCR2 mediates the angiogenic activity of ELR(+) CXC chemokines in a preclinical model of lung cancer.     

The Association of the Nm23-M1 Protein and β-Tubulin Correlates with Cell Differentiation

The Nm23 protein is a nucleoside diphosphate kinase (NDPK) and is thought to play a critical role in metastatic behavior. It has been reported that a NDPK activity is present in microtubules assembled in vitro. Since microtubule assembly is determinant in cell growth and differentiation, we investigated whether Nm23-M1 forms molecular complexes with β-tubulin in murine cells either actively proliferating or differentiating. For this purpose a polyclonal antibody against the GST-Nm23-M1 fusion protein was generated and employed to detect Nm23-M1/β-tubulin complexes in murine tumor cells derived from the Lewis lung carcinoma (3LL) and in undifferentiated and differentiated myogenic cells (C2C12). Immunoblotting and immunoprecipitation experiments performed using the anti-fusion protein antibody demonstrated that the Nm23-M1 protein is detectable in in vitro tumor cell lines and in in vivo primary tumors but not in spontaneous lung metastases. These data are in good agreement with data previously reported. Immunoprecipitation experiments demonstrated that the Nm23-M1 protein forms complexes with β-tubulin in in vitro tumor cell lines, but not in primary tumors. Furthermore, the Nm23-M1 protein forms complexes with β-tubulin in myogenic cells prior to and after differentiation. Interestingly, however, the level of the Nm23-M1/β-tubulin complexes is remarkably increased in differentiated myotubes. In conclusion, the results indicate that the Nm23-M1 protein forms molecular complexes with β-tubulin and that the number of complexes increases during the differentiation process of murine cells.     

Systemic treatment with interleukin-4 induces regression of pulmonary metastases in a murine renal cell carcinoma model

Advanced metastatic renal cell carcinoma has been shown to be responsive to immunotherapy but the response rate is still limited. We have investigated the therapeutic potential of systemic interleukin-4 (IL-4) administration for the treatment of pulmonary metastases in the murine Renca renal adenocarcinoma model. Renca cells were injected iv in Balb/c mice to induce multiple pulmonary tumor nodules. From Day 5, Renca-bearing mice were treated with two daily injections of recombinant murine IL-4 for 5 consecutive days. IL-4 treatment induced a significant reduction in the number of lung metastases in a dose-dependent manner and significantly augmented the survival of treated animals. Immunohistochemistry studies, performed on lung sections, showed macrophage and CD8⁺ T cell infiltration in the tumor nodules 1 day after the end of IL-4 treatment. The CD8 infiltration increased by Day 7 after IL-4 treatment. Granulocyte infiltration was not detectable. To clarify further the role of the immune system in IL-4 anti-tumor effect, mice were depleted of lymphocyte subpopulations by in vivo injections of specific antibodies prior to treatment with IL-4. Depletion of CD8⁺ T cells or AsGM1⁺ cells abrogated the effect of IL-4 on lung metastases, whereas depletion of CD4⁺ T cells had no impact. These data indicate that CD8⁺ T cells and AsGM1⁺ cells are involved in IL-4-induced regression of established renal cell carcinoma.