High collagenolytic activity in spontaneously highly metastatic variants derived from a human pancreatic cancer cell line (SUIT-2) in nude mice

Cell lines with high metastatic capacity to the lung were established by sequential passage of a human pancreatic cancer cell line (SUIT-2) through the lung of a nude mouse, via the lateral tail vein and from a subcutaneous inoculum. Cells of the parental SUIT-2 and sublines S2-VPx (x-cycle selection from SUIT-2 cells, by Vein-Pulmonary metastasis-culture) and S2-CPx (x-cycle selection, by Cutis-Pulmonary metastasis-culture) were injected intravenously or subcutaneously into nude mice to produce experimental or spontaneous lung metastasis. The S2-VP10 cell line produced pulmonary metastases in 100% of the nude mice, when injected intravenously. It failed, however, to produce more lung colonies than its parent cell line, when injected subcutaneously. The S2-CP8 cell line produced extensive pulmonary metastases in 100% of the nude mice, when injected either intravenously or subcutaneously. This study indicates that the nude mouse provided a good model for in vivo selection of metastatic cells from SUIT-2 cells both experimentally and spontaneously, and that the SUIT-2, S2-VPx, and S2-CPx cell lines will be valuable in the study of human cancer metastasis. We previously reported high levels of ezrin expression in the S2-VP10 and S2-CP8 cell lines. Here we show that these cell lines exhibit a greater capacity to invade or attach to various extracellular matrix components than the parent SUIT-2 cells. The S2-CP8 cell lines also exhibit greater level of type-I and type-IV collagen-degrading activity than the parent SUIT-2 cell line and the S2-VP10 cell line, which shows similar collagen-degrading activity to the parent SUIT-2 cells. In RT-PCR studies, SUIT-2, S2-CP8 and S2-VP10 cell lines constitutively expressed many matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP7, MMP-9, MMP-10 and MMP-14). These results suggest that some parameters that enhance adhesion and invasion are important to both experimental and spontaneous metastasis and the collagen degrading enzymes are predicted to play a key-role during spontaneous metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gene transduction of NK4, HGF antagonist,inhibits in vitro invasion and in vivo growth of human pancreatic cancer

In this study, we investigated the therapeutic effects of adenovirally-mediated transfer of the sequence of NK4, an antagonist for hepatocyte growth factor (HGF), against human pancreatic carcinoma. HGF has been implicated to play an important role in invasion and metastasis of various human cancers through tumor-stromal interactions. Although NK4 has been shown to block the metastatic behavior of cancer cells, problems with cellular delivery of NK4 must be addressed before it can be used for clinical trials. The effects of NK4 gene transduction mediated by recombinant adenovirus (Ad-NK4) were evaluated in a human pancreatic cancer cell line (SUIT-2) by in vitro scattering assays, invasion assays, and subcutaneous transplantation in nude mice. NK4 transduction markedly inhibited scattering and invasion of SUIT-2 cells stimulated by HGF without affecting cell proliferation in vitro. Furthermore, Ad-NK4 significantly inhibited the growth of tumors transplanted to nude mice. The tumor reduction induced by Ad-NK4 was associated with a decreased number of blood vessels surrounding the tumors. These findings suggest that Ad-NK4 gene therapy may be a unique and promising strategy for the treatment of pancreatic cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

可调控型反义基质金属蛋白酶9表达抑制人黑色素瘤细胞侵袭转移表型的研究

目的 探讨基质金属蛋白酶(MMP)-9与肿瘤转移的相关性。方法 利用基因重组技术构建反义MMP—9 cDNA四环素可调控型表达载体,用脂质体法转染反义MMP—9至转移性人黑色素瘤细胞株WM451(高表达MMP—9)。检测转染后细胞MMP—9表达水平的改变以及体外生长、侵袭、裸鼠体内成瘤及自发转移能力的变化。结果 转染反义基因后,WM451细胞MMP—9的表达及活性明显下降,同时MMP—2的表达也受到一定抑制,细胞生长速度、体外侵袭能力及棵鼠体内成瘤性及自发转移能力均受到一定程度抑制;运用四环素可以抑制四环素负调控逆转录病毒载体上的外源基因的表达。结论 反义MMP—9基因下调MMP—9的表达,可使人黑色素细胞转移能力受到一定程度的抑制,说明MMP—9在人黑色素瘤细胞转移过程中起重要作用。同时,四环素负调控逆转录病毒载体可以调控外源基因的表达。     

Correlation between spontaneous metastatic potential and type I collagenolytic activity in a human pancreatic cancer cell line (SUIT-2) and sublines

A human pancreatic cancer cell line (SUIT-2) and four sublines cloned in vitro (S2-007, S2-013, S2-020 and S2-028) were inoculated into nude mice for assessment of metastatic potentials. After 16 weeks of subcutaneous injection, the parent SUIT-2 line metastasized to the lungs and lymph nodes in three of six mice. S2-007 cells presented the highest metastatic potential in pulmonary (5/6) and lymph node (2/6) metastases among the four sublines. No metastasis was found in S2-028. The incidence of spontaneous pulmonary metastasis was correlated with that of pulmonary colonization after intravenous (i.v.) injection of cell clusters (r = 0.87, P = 0.056). Pulmonary colonization potential using single cells, however, did not always reflect a spontaneous metastatic ability. Type I collagenolytic activity in serum-free conditioned media of these cells was correlated effectively with the incidence of spontaneous pulmonary metastasis (r = 0.92, P = 0.026) and pulmonary colonization after i.v. injection of cell clusters (r = 0.95, P = 0.013). Thus, type I collagenolytic activity may possibly be essential to spontaneous cancer metastasis.     

Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor

Tumor microenvironment is crucial for cancer growth and progression as evidenced by reports on the significance of tumor angiogenesis and stromal cells. Using the HaCaT/HaCaT-ras human skin carcinogenesis model, we studied tumor progression from benign tumors to highly malignant squamous cell carcinomas. Progression of tumorigenic HaCaT-ras clones to more aggressive and eventually metastatic phenotypes was reproducibly achieved by their in vivo growth as subcutaneous tumors in nude mice. Their enhanced malignant phenotype was stably maintained in recultured tumor cells that represented, identified by chromosomal analysis, a distinct subpopulation of the parental line. Additional mutagenic effects were apparent in genetic alterations involving chromosomes 11 and 2, and in amplification and overexpression of the H-ras oncogene. Importantly, in vitro clonal selection of benign and malignant cell lines never resulted in late-stage malignant clones, indicating the importance of the in vivo environment in promoting an enhanced malignant phenotype. Independently of their H-ras status, all in vivo-progressed tumor cell lines (five of five) exhibited a constitutive and stable expression of the hematopoietic growth factors granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which may function as autocrine/paracrine mediators of tumor progression in vivo. Thus, malignant progression favored by the in vivo microenvironment requires both clonal selection of subpopulations adapted to in vivo growth and mutational events leading to stable functional alterations.     

Ectopic expression of MCAM/MUC18 increases in vitro motility and invasiveness,but decreases in vivo tumorigenesis and metastasis of a mouse melanoma K1735-9 subline in a syngeneic mouse model

Ectopic expression of MCAM/MUC18, a cell adhesion molecule in the immunoglobulin-like gene superfamily, induces two moMCAM/MUC18-minus, non-metastatic mouse melanoma K1735 sublines, K3 (tumor⁺/met^low) and K10 (tumor^?/met^low), to metastasize to lungs in a syngeneic C3H mouse model. In this report, we extended investigation of effects of moMCAM/MUC18 expression on tumorigenesis and metastasis in another lowly metastatic, however highly tumorigenic moMCAM/MUC18-minus mouse melanoma K1735 subline, K9 (tumor⁺⁺⁺/met^low). We transfected this subline with the moMCAM/MUC18 cDNA, selected for G418-resistant clones with different expression levels of moMCAM/MUC18, and used them for testing effects of MCAM/MUC18 expression on in vitro growth rate, motility, and invasiveness, in vivo subcutaneous tumor growth, and pulmonary metastasis in syngeneic C3H brown mice. Similar to K3 and K10 cells, increased expression of MCAM/MUC18 in K9 cells did not significantly affect in vitro growth rate, but increased in vitro motility and invasiveness. Surprisingly, increased expression of MCAM/MUC18 in K9 cells decreased their induction of tumorigenesis and suppressed their establishment of pulmonary nodules in syngeneic C3H brown mice. We concluded that increased MCAM/MUC18 expression in K9 subline increased in vitro epithelial-to-mesenchymal transition; however, it suppressed in vivo tumorigenicity and metastasis. Thus MCAM/MUC18 acts as a tumor and metastasis suppressor for the K9 subline, different from its role in other K1735 sublines, K3 and K10. Different intrinsic co-factors in different K1735 sublines, which modulate the functions of MCAM/MUC18 in the cells that interact differently to the tumor microenvironment, may render sublines manifest differently in tumorigenicity and metastasis in vivo.     

Immunogene therapy of murine fibrosarcoma using IL-15 gene with high translation efficiency.

We have recently found that translational efficiency is up-regulated by an alternative exon in IL-15 mRNA in mice. In a malignancy model using BALB/c mice and syngeneic Meth A fibrosarcoma (Meth A), we successfully applied immunological gene therapy with IL-15 protein using alternative IL-15 cDNA with high translational efficiency. Two expression vectors carrying the murine IL-15 gene were constructed for use in tumor immunotherapy, one utilizing IL-15 cDNA with alternative exon 5 and the second utilizing IL-15 cDNA with normal exon 5. The first vector induced the production of a large amount of IL-15 protein in Meth A cells, whereas tumor cells transfected by the second vector produced only a marginal level of IL-15 protein. Although cell growth of both transfectants in vitro remained unchanged, inoculation of clones transfected with normal IL-15 cDNA resulted in progressive tumor growth, while clones transfected with alternative IL-15 cDNA led to the rejection of the tumor. The clone producing high levels of IL-15 grew progressively in nude mice and mice treated with anti-CD4 monoclonal antibodies (mAb), whereas the growth of the transfectants was retarded in anti-CD8 mAb- or anti-asialo GM1 antibody-treated mice. Cured mice were shown to have generated immunity against a subsequent challenge with the wild type of Meth A but not against Meth 1 tumor cells, another type of fibrosarcoma derived from BALB/c mice. Thus, tumor therapy based on IL-15 gene transfection was effective against Meth A tumor cells, suggesting a possible application to human neoplasms.     

MicroRNA-17-3p is a prostate tumor suppressor in vitro and in vivo,and is decreased in high grade prostate tumors analyzed by laser capture microdissection

MicroRNAs (miRs) are a novel class of RNAs with important roles in regulating gene expression. To identify miRs controlling prostate tumor progression, we utilized unique human prostate sublines derived from the parental P69 cell line, which differ in their tumorigenic properties in vivo. Grown embedded in laminin-rich extracellular matrix (lrECM) gels these genetically-related sublines displayed drastically different morphologies correlating with their behaviour in vivo. The non-tumorigenic P69 subline grew as multicellular acini with a defined lumen and basal/polar expression of relevant marker proteins. M12, a highly tumorigenic, metastatic derivative, grew as a disorganized mass of cells with no polarization, whereas the F6 subline, a weakly tumorigenic, non-metastatic M12 variant, reverted to acini formation akin to the P69 cell line. These sublines also differed in expression of vimentin, which was high in M12, but low in F6 and P69 sublines. Analysis of vimentin’s conserved 3′-UTR suggested several miRs that could regulate vimentin expression. The lack of miR-17-3p expression correlated with an increase in vimentin synthesis and tumorigenicity. Stable expression of miR-17-3p in the M12 subline reduced vimentin levels 85% and reverted growth to organized, polarized acini in lrECM gels. In vitro motility and invasion assays suggested a decrease in tumorigenic behaviour, confirmed by reduced tumor growth in male athymic, nude mice dependent on miR-17-3p expression. Analysis of LCM-purified clinical human prostatectomy specimens confirmed that miR-17-3p levels were reduced in tumor cells. These results suggest that miR-17-3p functions as a tumor suppressor, representing a novel target to block prostate tumor progression.     

不同组织微环境中人胃癌异种移植瘤侵袭性及基质金属蛋白酶谱表达的差异

目的 探讨组织微环境对癌细胞侵袭性影响机制中基质金属蛋白酶表达的意义。方法 取人胃腺癌组织移植于裸小鼠皮下,成瘤后进行皮下和腹腔内传代接种,形态学观察2处异种移植瘤侵袭性的不同并用免疫组织化学链霉素抗生物素蛋白-过氧化物酶法检测基质金属蛋白酶(MMP)-2、MMP-7、MMP-9、MMP-13、TM1-MMP、TM2-MMP、TM3-MMP 7种MMPs在瘤组织中的表达。结果 人胃癌裸小鼠皮下异种移植瘤呈膨胀性生长,侵袭性不明显;除MMP-7外,其他6种MMPs在皮下移植瘤细胞及间质中均无表达。腹腔内移植瘤呈侵袭性生长、纤维间质增多,多种MMPs均在侵袭前沿的肿瘤细胞及间质中表达。同一瘤株来源的人胃癌细胞在裸小鼠不同组织环境中所呈现的侵袭性及MMPs表达差异均有显著性。结论 (1)肿瘤细胞与相邻的间质细胞之间存在相互诱导作用,组织环境对肿瘤侵袭表型可有决定性的影响。(2)MMPs的表达与肿瘤细胞生长方式及侵袭性有密切联系;肿瘤侵袭前沿的间质细胞产生的MMPs也可能参与肿瘤细胞的侵袭过程。     

Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.     

Growth and metastatic behavior of human tumor cells implanted into nude and beige nude mice

The growth and metastatic behavior of several human tumor lines grown in adult nude mice, nude mice pretreated with antiserum against asialo GM1 glycoprotein, and beige nude mice were studied. The cell lines were all injected s.c. and i.v. A human colon carcinoma line was also injected into the spleen, and two human renal carcinoma lines were injected into renal subcapsule. All the tumor lines grew as fast or faster in adult nude mice compared with beige nude mice. There were no discernible differences in the production of experimental lung metastasis among the three groups of mice, but human colorectal carcinoma cells and human renal carcinoma cells produced more metastases in nude mice than in beige nude mice after intrasplenic or renal subcapsule injection, respectively. In vitro cytotoxicity assays confirmed that adult nude mice had high levels of natural killer (NK) cell activity whereas nude mice pretreated with anti-asialo GM1 serum and beige nude mice did not. The in vitro NK cell activity of nude mice was demonstrable against mouse lymphoma cells but not against human leukemia cells which were sensitive to lysis by human NK cells. These results suggest that the implantation of human tumor cells into beige nude mice, which are deficient in NK cell activity does not provide an advantageous model for the study of growth and metastasis of human neoplasms.Abbreviations NK natural killer - HCC human colon carcinoma - HRCC human renal cell carcinoma - anti-asGM1 anti-asialo GM1 - i.s. intrasplenic - RSC renal subcapsule - HBSS Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution     

裸鼠荷人鼻咽癌细胞株后免疫状态的动态观察

本实验选用4～6周龄NC系裸鼠,原代用双侧颈背部皮下接种CNE-2细胞株,鼠间传代用套针双侧接种上代的新鲜瘤块,共传8代,成功率100%。动态观测裸鼠荷瘤后免疫状态变化的结果如下:(1)净体重显著减轻;(2)脾系数增大,瘤重增加,两者间高度正相关(r=0.8395);(3)脾NK活性在第5天时轻度升高,10天时开始下降,15天后显著下降;NK活性与瘤重及脾系数均呈高度负相关(r_1=-0.7132,r_2=-0.8237);(4)第5天时,脾NK细胞体外对人干扰素反应性良好,10天时仍有一定的反应性,15天后失去反应性;(5)荷瘤裸鼠牌细胞自然转化率高于对照组,而对ConA刺激的反应性低于对照组;(6)“癌克星”通过降低血清MDA、提高NK活性及增加瘤组织中单核细胞浸润而起抑瘤生长的作用。揭示在T细胞缺陷的裸鼠,其脾NK细胞是抗移植瘤生长的重要免疫活性细胞。     

转染IL-21的CD34~+脐血造血干细胞抗裸鼠卵巢癌作用研究

目的 探讨IL-21转染的脐血造血干细胞(CD34~+UBSC·IL-21)对荷卵巢癌裸鼠的治疗作用.方法 从脐血分离CD34~+造血干细胞,体外培养扩增后用于重组体pIRES2-IL-21-EGFP转染.以肿瘤大小、荷瘤鼠生存期判断CD34~+UBSC-IL-21对荷瘤裸鼠的治疗效应.以RT-PCR、免疫荧光、ELISA、Western blot、脾细胞增殖试验及免疫组化法分别鉴定CD34~+UBSC和肿瘤组织中IL-21的表达及活性.裸鼠脾细胞中NK细胞含量及脾细胞的杀伤效应、血清中IFN-γ和TNF-α水平分别用FCM与ELISA检测.结果 pIRES2-IL-21-EGFP成功转染CD34~+UBSC.CD34~+UBSC-IL-21能抑制肿瘤生长,延长荷瘤裸鼠生存期,治疗鼠肿瘤局部能表达IL-21、血清IFN-γ和TNF-α水平升高,NK细胞含量及NK细胞杀伤活性明显增强,与其他组相比,差异有统计学意义(P<0.01).结论 转染IL-21的CD34~+UBSC有良好的抗裸鼠卵巢癌作用,该结果为临床使用UBSC为载体的基因治疗卵巢癌研究奠定了基础.     

Phenotypic and functional characterization of a panel of cytotoxic murine NK cell clones that are heterogeneous in their enhancement of Ig secretion in vitro

NK cells not only function as cytotoxic effector cells, but also have immunoregulatory roles including the enhancement of Ig secretion. To have a stable and uniform population of NK cells to study their role in Ig secretion, we generated murine NK clones. Thus, culture of splenocytes from mice that were homozygous for a mutation in the p53 tumor suppressor gene (p53-KO) with IL-2 and poly(IC) resulted in a long-term NK line, from which four stable clones were derived. This approach also yielded a long-term NK line from splenocytes of normal C57BL/6 mice. Identification of the clones as members of the NK lineage was based on large granular morphology, expression of NK-TR and absence of TCR gene rearrangement. Flow cytometry revealed that all clones expressed IL-2R alpha and beta, chains and B220, but no CD3, NK1.1, DX5 or Ly-49. RT-PCR analysis showed heterogeneity in NK1.1 gene expression, and demonstrated expression of perforin and several granzymes in all clones. Three out of four clones lysed YAC-1, but not P815 target cells, corresponding to a pattern of NK specificity. All NK clones enhanced Ig secretion in an in vitro model for T cell- independent type 2 antigens, albeit to varying degrees. We found no correlation between the degree of helper activity of the NK clones and the level of their cytotoxic activity on YAC-1 targets. Thus, we established murine NK clones, and show that they mediate both cytotoxicity and enhancement of Ig secretion.      

Neem (Azadirachta indica) leaf preparation induces prophylactic growth inhibition of murine Ehrlich carcinoma in Swiss and C57BL/6 mice by activation of NK cells and NK-T cells

We have reported earlier that pretreatment of mice with neem leaf preparation (NLP) causes prophylactic growth inhibition of murine Ehrlich's carcinoma (EC) and B16 melanoma. Using adoptive cell transfer technology, here we have established that NLP-mediated activation of immune cells may be involved in tumor growth restriction. Mononuclear cells from blood and spleen of NLP-activated Swiss and C57BL/6 mice causes enhanced cytotoxicity to murine EC cells in vitro. Fractionation of spleen cells exhibited greater percentage of tumor cell lysis in macrophage and B-cell-depleted NK and T-cell-rich fractions. Flow cytometric analysis revealed in both blood and spleen, NK cells (DX5+ or NK1.1+) and NK-T cells (CD3+/DX5+ or CD3+/NK1.1+) were increased in number in Swiss, C57BL/6 and athymic nude mice after pretreatment with NLP. NLP-stimulated spleen cells showed greater secretion of TNFalpha and IFNgamma. Thus, NLP-activated NK and NK-T cells in mice may regulate tumor cell cytotoxicity by enhancing the secretion of different cytotoxic cytokines.     

Thy-1-positive dendritic epidermal cells contain a killer protein perforin

The killer cell characteristics of Thy-1-positive dendritic epidermal cells (Thy-1+ DEC) were examined. Four Thy-1+ DEC clones which were established from athymic nude mice exhibited spontaneous or lectin-redirectable cytotoxic activity against some murine tumor cell lines in a 4 h 51Cr-release assay. A colorimetric assay for benzyloxycarbonyl-L-lysine-thiobenzyl ester esterase revealed a strong serine esterase activity expressed in all cell clones. In addition, Northern blot analysis using a murine perforin cDNA probe revealed that all four Thy-1+ DEC clones expressed abundant mRNA for perforin, as do most killer T cells. More importantly, immunocytochemical staining with an anti-perforin monoclonal antibody revealed that not only all four Thy-1+ DEC clones but also a part of freshly isolated Thy-1+ DEC from normal and nude mice contained perforin. These results demonstrate that Thy-1+ DEC exhibit typical killer cell characteristics in vitro and in vivo. These data also suggest that Thy-1+ DEC may play a cytotoxic role in protecting the integrity of skin from infection or neoplastic transformation.     

Tumorigenic potential of extracellular matrix metalloproteinase inducer

Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs.     

Retroviral IFN-alpha gene transfer combined with gemcitabine acts synergistically via cell cycle alteration in human pancreatic carcinoma cells implanted orthotopically in nude mice.

Standard chemotherapy for pancreatic carcinoma is based on the use of gemcitabine. The clinical benefit of interferon-alpha (IFN-alpha) in advanced pancreatic cancer has been shown. However, it has been demonstrated that to be effective, there is a need for a constant amount of IFN-alpha at the site of the tumor. Therefore, we examined transfection of the human pancreatic cancer cell line DAN-G with a retrovirus encoding for IFN-alpha and the effect of IFN-alpha gene expression alone or in combination with gemcitabine on growth inhibition of DAN-G pancreatic cancer cells in vitro and in vivo in orthotopically implanted DAN-G cells in nude mice. DAN-G cells could be efficiently transfected retrovirally by the human IFN-alpha gene and significantly enhanced the levels of IFN-alpha mRNA. In vitro gemcitabine led to an alteration of G1/S phase progression in transduced as well as untransduced cells, whereas IFN-alpha led to a significant decrease in cell viability in the transduced cells via delay in the progression of the S phase but no alteration of G1/S phase progression. In vivo, tumor volume in mice was reduced significantly with gemcitabine combined with IFN-alpha (76% +/- 8.3%) compared with gemcitabine alone (62.9% +/- 7.3%) or IFN-alpha alone (24.4% +/- 5.2%) compared with untreated animals. We conclude that gemcitabine and IFN-alpha concomitantly inhibited tumor cell proliferation significantly.     

Promotion of cell proliferation and inhibition of ADCC by cancerous immunoglobulin expressed in cancer cell lines

To explore the significance of cancerous immunoglobulin (Ig) in cancer cell growth, HeLa cervical cancer cells were stably transfected with small interfering RNA (siRNA) that specifically, efficiently and consistently silences the expression of heavy chain genes of all immunoglobulin isotypes. This stable cell line was used to examine cell viability, colony formation and tumor growth in athymic nude mice. The results of these experiments indicated that siRNA-mediated knockdown of cancerous Ig inhibited cell growth in vitro and suppressed tumor cell growth in immune-deficient nude mice in vivo. Similarly, this siRNA also inhibited the growth of MGC gastric cancer cells and MCF-7 breast cancer cells. Furthermore, the presence of cancerous Ig specifically reduced antibody-dependent cell-mediated cytotoxicity (ADCC) induced by an anti-human epithelial growth factor receptor (EGFR) antibody in a dose-dependent manner, suggesting that the cancerous Ig-Fc receptor interaction inhibits natural killer cell (or NK cell) effector function. The prevalent expression of Ig in human carcinomas and its capacity to promote growth and inhibit immunity might have important implications in growth regulation and targeted therapy for human cancers.     

NK4基因转染对裸鼠人淋巴瘤移植瘤的抑制作用

目的 探讨NK4基因转染对裸鼠人淋巴瘤移植瘤的抑制作用及其机制.方法 采用NK4基因重组质粒pVITRO2-NK4转染的Raji细胞建立裸鼠皮下人淋巴瘤移植瘤模型,动态监测裸鼠体重和肿瘤大小.8周后获取瘤组织,分别采用免疫组化和脱氧核糖核酸末端转移酶介导的缺口末端标记法(TdT-mediated dUTP nick end labeling,TUNEL)检测移植瘤组织的细胞凋亡和微血管密度(microvessel density,MVD),并进行相关分析.结果 NK4基因转染组裸鼠移植瘤体积明显小于对照组(P<0.01),而对照组间差异无显著性(P>0.05);各组间裸鼠体重降低,差异无显著性(P<0.01).NK4基因转染组的淋巴瘤细胞AI值达237±10.94,而质粒pVITRO2转染组和未转染Raji组的AI值分别为79.7±30.8和81.7±22.2,NK4基因转染组明显高于对照组(P<0.001),而对照组间差异无显著性(P>0.05);NK4基因转染组MVD为4.7±1.52,而质粒pVITRO2转染组和未转染Raji组MVD分别为12.0±1.00和10.66±1.53,NK4基因转染组明显低于对照组(P<0.001),而对照组间差异无显著性(P>0.05).结论 NK4基因转染可明显抑制裸鼠人淋巴瘤移植瘤的生长,其可能通过抑制肿瘤血管新生和促肿瘤细胞凋亡而发挥其效应.