Retroviral gene transfer induced constitutive expression of interleukin-2 or interferon-gamma in irradiated human melanoma cells.

Cytokines are important modulators of host antitumor responses. Two of these cytokines, interleukin-2 (IL-2) and interferon gamma (IFN-gamma), are produced after antigen-induced activation of helper lymphocytes. The cytokines are released into the immediate vicinity where they either interact with the appropriate receptors on effector cell populations or are rapidly degraded. To mimic this physiologic release of cytokines at the effector-target site, we used retroviral vectors to transduce melanoma cells with the IL-2 or IFN-gamma cDNA. Five melanoma cell lines were transduced with IL-2- or IFN-gamma-containing vectors and secreted IL-2 at 1 to 40 U/mL/10(6) cells/24 h or IFN-gamma 1 to 8 U/mL/10(6) cells/24 h, respectively. After gamma irradiation, these cells continued to secrete cytokines for about 3 to 4 weeks. Secretion of IFN-gamma induced upregulation of major histocompatibility complex class I molecules in a subset of melanoma cell lines. IL-2 production by human melanoma xenografts induced tumor rejection in BALB/c nu/nu mice, showing the in vivo effect of this cytokine. This study shows that (1) human melanoma cells can be stably transduced with cytokine-containing retroviral vectors; (2) cytokines are secreted constitutively by the transduced tumor cells and have the expected biologic effects in vitro and in vivo; and (3) after gamma irradiation, cytokines continue to be secreted for several weeks. These data suggest that irradiated cytokine-secreting allogenic or autologous tumor cells can be used in vaccination protocols for cancer patients.     

Recombinant human interleukin-4 inhibits growth of some human lung tumor cell lines in vitro and in vivo

Cytokines play an important role in activating the immune system against malignant cells. One of these cytokines, interleukin-4 (IL-4) has entered clinical phase I trials because of its immunoregulatory potency. In the present study we report that recombinant human (rh) IL- 4 has major direct antiproliferative effects on one human lung cancer cell line (CCL 185) in vitro as measured by a human tumor cloning assay (HTCA), tritiated thymidine uptake, and counting cell numbers and marginal activity in a second cell line (HTB 56) in the HTCA. This activity could be abolished by neutralizing antibody against rhIL-4. The biological response of the tumor cells to the cytokine is correlated with expression of receptors for human IL-4 on both the mRNA level and the protein level. The responsive cell line, CCL 185, secretes IL-6 after being incubated with rhIL-4. On the other hand, neutralizing antibodies against IL-6 showed no influence on the growth modulatory efficacy of rhIL-4 in this cell line. Furthermore, CCL 185 does not show detectable production of IL-1, tumor necrosis factor alpha or interferon gamma after incubation with rhIL-4. Thus, the response to rhIL-4 is not mediated through autocrine production of these cytokines triggered by rhIL-4. In a next series of experiments some of the cell lines were xenotransplanted to BALB/c nu/nu mice. Subsequently, the mice were treated for 12 days with two doses of 0.5 mg/m2 rhIL-4 or control vehicle subcutaneously per day. Treatment with rhIL-4 yielded a significant inhibition of tumor growth versus control in two of the non-small cell lung cancer cell lines being responsive in vitro (CCL 185, HTB 56). Histology of the tumors in both groups showed no marked infiltration of the tumors with murine hematopoietic and lymphocytic cells consistent with the species specificity of IL-4. In contrast, no tumor growth inhibition was found in the small cell lung cancer cell lines (HTB 119, HTB 120) being nonresponsive in vitro. We conclude that rhIL-4 has direct antiproliferative effects on the growth of some human non-small cell lung cancer cell lines in vitro and in vivo, which together with its regulatory effects on various effector cell populations makes this cytokine an interesting candidate for further investigation in experimental cancer treatment.     

Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene

Malignancies arising from retrovirally transduced hematopoietic stem cells have been reported in animal models and human gene therapy trials. Whether mature lymphocytes are susceptible to insertional mutagenesis is unknown. We have characterized a primary human CD8(+) T-cell clone, which exhibited logarithmic ex vivo growth in the absence of exogenous cytokine support for more than 1 year after transduction with a murine leukemia virus-based vector encoding the T-cell growth factor IL-15. Phenotypically, the clone was CD28(-), CD45RA(-), CD45RO(+), and CD62L(-), a profile consistent with effector memory T lymphocytes. After gene transfer with tumor-antigen-specific T-cell receptors, the clone secreted IFN-gamma upon encountering tumor targets, providing further evidence that they derived from mature lymphocytes. Gene-expression analyses revealed no evidence of insertional activation of genes flanking the retroviral insertion sites. The clone exhibited constitutive telomerase activity, and the presence of autocrine loop was suggested by impaired cell proliferation following knockdown of IL-15R alpha expression. The generation of this cell line suggests that nonphysiologic expression of IL-15 can result in the long-term in vitro growth of mature human T lymphocytes. The cytokine-independent growth of this line was a rare event that has not been observed in other IL-15 vector transduction experiments or with any other integrating vector system. It does not appear that the retroviral vector integration sites played a role in the continuous growth of this cell clone, but this remains under investigation.     

Transduction of human hepatocellular carcinoma cells with human alpha-interferon gene via retroviral vector

AIM:To investigate the therapeutic potential of gamma interferon (IFN-alpha) genemodified human hepatocellular carcinoma (HCC) cells.METHODS:The IFN-alpha gene was introduced retrovirally into four HCC cell lines.Secreted IFN-alpha activity was assessed using bioassay. The expression of MHC molecules was detected by FACS.Tumorigenicity was analysed by tumor formation in nude mice.RESULTS:Four IFN-alpha gene transduced HCC cell lines secreted different amounts of IFN-alpha, as in the same case of five clones derived from one HCC cell line. Transduction with IFN-alpha caused significant increase in the expression of major histocompatibility complex (MHC) antigens on HCC cells. The expression of HLA class I was increased by 2-3 times in terms of mean fluorescence intensities, while for class II expression, the percentage of positive cells augmented from < 10% to &lg 50%. When equal amount of tumor cells were injected into nude mice, the tumor igenicity some transduced cells decreased dramantically.CONCLUSION:IFN-alpha gene transduction can convert weakly imunogenic HCC cells to activate antitumor immune response, and further pave the way for the future use of such gene modified tumor cells as a modality for the cancer immunotherapy.     

腺病毒介导多基因对肝癌细胞凋亡的诱导作用

目的 研究腺病毒介导的多基因（ｐ５３、Ｂ７－１、ＧＭ－ＣＳＦ、ＩＬ－２）对肝癌细胞系调亡的诱导,及对肝癌细胞系裸鼠体内成瘤性改变的影响。方法 应用光镜、电镜和ＴＵＮＥＬ法,检测肝癌细胞系感染腺病毒介导的多基因后凋亡的发生,检测ＨｅｐＧ２细胞导入腺病毒多基因后裸鼠体内的成瘤性改变。结果 肝癌细胞系导入多基因后发生凋亡,并对化疗药物顺铂的敏感性增加,册时给予１０ｍｇ／Ｌ的顺铂,可使近３０％的肝癌细胞发     

腺病毒介导MDA-7/IL-24选择性促进肝癌细胞的凋亡和增殖阻滞

目的 观察MDA-7／IL-24基因对不同p53状态人肝癌细胞HepG2、MHCC97L以及Hep3B和正常的肝细胞L02的选择性杀伤作用，为肝癌的基因治疗提供理论基础。方法 将携带人MDA-7／IL-24基因的腺病毒Ad．mda-7感染人正常肝细胞L02和不同p53状态的肝癌细胞HepG2，MHCC97L、Hep3B。通过逆转录聚合酶链反应方法观察MDA7／IL24基因的表达，酶联免疫吸附法检测细胞培养上清液中MDA-7／IL-24蛋白的浓度，通过四甲基偶氮唑盐染色法及Hoechst染色观察MDA-7／IL-24对肝癌细胞的生长抑制和杀伤作用，Annexin-V和碘化丙啶双染后流式细胞仪检测细胞的凋亡，应用流式细胞仪检测细胞周期。结果 复制缺陷型腺病毒能介导外源基因MDA-7／IL-24在肝癌细胞株HepG2，MHCC97L和Hep3B以及正常细胞L02中高效表达。细胞培养上清液中有MDA-7／IL-24蛋白表达。MDA-7／IL-24能明显抑制各种肝癌细胞的生长，Hoechst染色提示MDA-7／IL-24促进肝癌细胞的凋亡，流式细胞仪提示MDA-7能选择性杀伤肝癌细胞而对正常的肝细胞无影响，细胞周期分析提示MDA-7／IL-24阻滞肝癌细胞在G2／M期，同时对正常的肝细胞没有促凋亡作用和增殖阻滞作用。结论 复制缺陷型重组腺病毒载体Ad．mda-7能介导MDA-7／IL-24基因在人肝癌细胞中高效表达，选择性地杀伤肝癌细胞HepG2、MHCC97L和Hep3B，促进细胞增殖阻滞及诱导肿瘤细胞凋亡而与肿瘤细胞的P53基因的状态无关，同时对正常的肝细胞L02无任何毒性作用。     

Interleukin-15 blocks apoptosis and induces proliferation of the human myeloma cell line OH-2 and freshly isolated myeloma cells

The growth factor-dependent myeloma cell line OH-2, which has previously been shown to be responsive to interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and lymphotoxin, was examined for response to other growth factors. Enhanced proliferation was found in the presence of IL-10, IL-15, IL-2 and insulin growth factor (IGF)-1. Proliferation was strongest in response to IL-6, intermediate and roughly equipotent in response to IL-15, IL-10 and TNF-alpha, and modest in response to IL-2 and IGF-1. IL-15 was synergistic with TNF-alpha, whereas combinations of IL-15 and the other cytokines were merely additive. IL-15-induced proliferation could not be blocked by neutralizing antibody against gp 130, the common transducer chain of IL-6 and related cytokines. IL-15 and IL-6 prevented apoptosis equally well, both better than TNF-alpha, IL-10, and IGF-1. In four out of six samples of purified primary cells, IL-15 and IL-6 induced proliferation. Furthermore, IL-15 mRNA was detected by RT-PCR in most myeloma cell lines and freshly isolated purified patient samples. IL-15 protein was detectable only in one out of about 20 tested cell supernatants from patients and myeloma cell lines. The OH-2 cell line is multi-responsive to cytokines and is a good system for the study of integration of cytokine signal transduction and growth control in myeloma. IL-15 represents a novel modality of growth regulation in myeloma.     

gamma-c gene transfer into SCID X1 patients' B-cell lines restores normal high-affinity interleukin-2 receptor expression and function

SCID X1 is characterized by faulty T-cell and natural killer cell differentiation caused by mutation of the gamma-c chain gene encoding a number of multiple cytokine receptors (interleukin-2 [IL-2], IL-4, IL- 7, IL-9, and IL-15 receptors). To assess the feasibility of inducing long-term expression and function of the gamma-c chain, Epstein-Barr virus (EBV)-transformed B-cell lines from two patients with SCID X1 were transduced with a Moloney-derived retroviral vector containing the gamma-c chain cDNA. The viral LTR was used as the promoter. Immediately after two cycles of coculture with the psi-crip clone producing the MFG(B2)-gamma-c cDNA vector, gamma-c expression, assessed by detection of the mRNA and membrane protein expression, was found in 15% to 20% of cells. The degree of membrane expression was similar to that in control EBV-B cells. Expression increased steadily over 6 months, becoming detectable in 100% of cells, and remained stable thereafter for a total of 9 months, reflecting positive selection of transduced cells. A study of provirus integration sites showed multiple integration. The expressed gamma-c was functional, because it restored high-affinity IL- 2 receptor binding, IL-2 endocytosis, and IL-2-triggered phosphorylation of JAK-3 tyrosine kinase. Similar results were obtained with the two B-cell lines. These results show that efficient gamma-c gene transfer into B-cells lacking functional gamma-c is feasible and results in strong and stable expression of a functional gamma-c chain, apparently conferring a selective growth advantage in culture. Further in vitro studies of gamma-c gene transfer into gamma-c- hematopoietic progenitors are being conducted to assess the feasibility of correcting lymphocyte differentiation defects.     

Retrovirus-mediated IL-7 expression in leukemic dendritic cells generated from primary acute myelogenous leukemias enhances their functional properties

Myeloid lineage-derived dendritic cells (DCs) are considered the professional antigen-presenting cell type responsible for eliciting T-cell-mediated immune responses. Acute myelogenous leukemia (AML) is a disease in which tumor antigens are expressed by the malignant clone that also has the potential to differentiate into DC-like cells (leukemic DCs) with antigen-presenting capacity. This study investigated whether the constitutive expression of the cytokine interleukin-7 (IL-7) in primary AML cells during their differentiation toward leukemic DCs results in superior antigen-presenting cells. A bicistronic retroviral vector encoding the IL-7 cytokine and the surface immunoselectable low-affinity nerve growth factor receptor (LNGFr) gene was constructed and used for transduction experiments. A serum-free system was used to transduce and differentiate leukemic cells toward leukemic DCs. The study included 8 patients with AML. The transduction efficiency with the cytokine vector varied among patients, ranging from 5% to 30% as judged by LNGFr expression. The leukemic origin of the transduced cells was confirmed in a patient with a chromosomal translocation t(9:11) by fluorescence in situ hybridization analysis. Cytokine modified-cells consistently secreted IL-7 (mean, 415 pg +/- 190/10(6) cells/48 hours; n = 5). We demonstrate that IL-7-transduced cells are included in the differentiated leukemic DC subset, and, as shown in a particular case, that about half of the mature CD80(+) and CD83(+) populations coexpress the LNGFr transgene. In addition, IL-7-modified leukemic cells induce stronger allo-T-cell stimulation and higher amounts of IL-2 production in T cells compared with control groups. Finally, cytokine-transduced leukemic DCs can effectively prime and generate cytotoxic T lymphocytes against autologous leukemic blasts.     

白细胞介素—2基因转导入胃癌细胞的实验研究

为研究白细胞介素－２基因转导人胃癌细胞的可行性和有效性，用直人核表达载体，将人ＩＬ－２ｃＤＮＡ转导入人胃产吕细胞株ＳＧＣ－７９０１细胞表达，并观察了基因转导和表达对胃癌细胞体外生长特性和ＬＡＫ细胞的肿瘤杀伤作用的影响，以及基因转导细胞的体内致瘤性。     

Role of interleukin-6 in the proliferation of human multiple myeloma cell lines OCI-My 1 to 7 established from patients with advanced stage of the disease

Interleukin-6 (IL-6) has been shown to stimulate the proliferation of multiple myeloma cells purified to a high degree from human bone marrow. IL-6 production in multiple myeloma has been attributed to cells belonging to the myeloma clone, thus supporting a mechanism of autostimulation. In addition, it has been shown that IL-6 may be produced by auxiliary cell populations of the bone marrow that are not part of the myeloma clone. A definitive separation of both putative sources for IL-6 may be difficult to achieve in fresh patient IL-6 growth requirement and production by pure myeloma cell populations using seven human myeloma cell lines (OCI-My 1 to 7) that were established from patients with advanced disease. The proliferative response of each line to recombinant IL-6 was measured in a clonogenic assay providing human plasma and methylcellulose as a viscous support and by 3H-thymidine uptake in liquid suspension culture. We observed marked heterogeneity, ranging from IL-6-dependent colony formation by OCI-My 4, to IL-6-independent growth. All lines expressed mRNA for the IL-6 receptor. Expression of IL-6 mRNA was analyzed after amplification by polymerase chain reaction and was present in five of seven lines. IL-6 protein was detected by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of two lines (OCI-My 3 and 2). Its functional activity was confirmed in a bioassay using the IL-6-dependent murine hybridoma line B 13.29. This activity was neutralized by anti-IL-6 antibody. Two lines did not express mRNA for IL-6. The remaining three lines expressed mRNA for IL-6, but did not secrete IL-6 protein. Immunoprecipitation experiments with lysates of one of these three lines did not detect the presence of IL-6 protein. These results suggest that autocrine stimulation by IL-6 may occur in some cell lines derived from patients with multiple myeloma. However, it does not represent a universal mechanism in myeloma cell growth.     

Effects of phorbol esters on an interleukin-3-dependent cell line

FDC-P1 is an interleukin-3 (IL-3)-dependent cell line that ceases to proliferate in the absence of IL-3. We have isolated variant cell lines from FDC-P1 that grow in response to phorbol myristate acetate (PMA). These variant cell lines (FD/PMA) have maintained their PMA-dependency for over 1 year. Lymphokine gene expression, which would support growth, was not detected in FD/PMA lines. FD/PMA lines had a different cell surface phenotype than the parental line. Mac-1, Mac-2, and Mac-3 were readily detected on the cell surface of FD/PMA lines; however, these antigens were not detected on FDC-P1. Because protein kinase C (PKC) activation may mediate PMA effects, we examined this kinase. PKC activity quantitated by 32P-incorporation into histone was increased in FDC-P1 as compared with FD/PMA cultured in IL-3. Moreover, PKC activity was undetectable in FD/PMA lines cultured in PMA. Using Western blotting, immunoreactive PKC was readily detected in cytosolic and solubilized particulate fractions of FDC-P1 cells, not but in FD/PMA cell extracts. Comparisons between the parental and FD/PMA lines should provide insight into IL-3- and PMA-mediated signal transduction.     

基因枪转导K-ras突变反义基因对人胰腺癌细胞生长曲线的影响

目的探讨基因枪转导K-ras突变反义基因对胰腺癌细胞生长的影响。方法利用基因枪技术,将反义基因转导入宿主细胞,通过活细胞计数试剂盒Cell Counting Kit-8和Thermo Multiskan Ascant酶标仪观察K-ras突变特异性反义基因转导后对AsPC-1、MiaPaCa-2和BxPC-3三种人胰腺癌细胞的生长曲线变化情况。结果转导K-ras突变特异性反义基因后,突变型AsPC-1和MiaPaCa-2胰腺癌细胞的生长曲线明显向右下移动。结论转导K-ras突变特异性反义基因后,具有突变型K-ras基因的胰腺癌细胞系的生长都受到明显抑制。     

Tissue-specific expression of herpes simplex virus thymidine kinase gene delivered by adeno-associated virus inhibits the growth of human hepatocellular carcinoma in athymic mice

About 70% of hepatocellular carcinomas are known to express α-fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the α-fetoprotein enhancer and albumin promoter. We previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells in vivo. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus in vivo, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of in vivo transduction of tumor cell with rAAV.     

HIV-1 envelope protein gp41 modulates expression of interleukin-10 and chemokine receptors on monocytes, astrocytes and neurones

OBJECTIVE: To analyse the effect of HIV-1 transmembrane protein gp41 on cytokine production and chemokine receptor expression in blood and brain. DESIGN: Because previous results had demonstrated that recombinant gp41 contributes to HIV-induced dysfunction of blood immune cells we investigated its effect on interleukin (IL)-10 synthesis and expression of the HIV coreceptors CCR5 and CXCR4 in different human brain cells. METHODS: Astrocytic, microglial and neuronal cell lines were incubated with the extracellular domain of gp41 (aa565-647). Secretion of IL-10 into the medium was measured by ELISA. Chemokine receptor expression was analysed by fluorescence activated cell sorting and by RT-PCR. RESULTS: Incubation of the astrocytic cell line U87 with gp41 induced more than a 10 fold up-regulation of IL-10 secretion. This modulation was shown to be time- and dose-dependent. Use of inhibitors for different signal transduction pathways indicated a similar transduction cascade for the alteration of IL-10 production in astrocytes as in monocytes with participation of cAMP/adenylate cyclase and activation of p70S6 kinase. To a lesser extent IL-10 synthesis was also up-regulated by gp41 in the neuronal cell line SK-N-SH. In all cell types up-regulation of IL-10 paralleled by an enhanced expression of the chemokine receptor and HIV-1 coreceptor CCR5. This up-regulation was driven by IL-10 as shown by use of an IL-10 antibody. Expression of the chemokine receptor CXCR4 was only slightly altered. CONCLUSIONS: These findings suggest a role for gp41 in the modulation of brain-specific host defence, cell migration and cell infectivity by HIV.     

Expression of human glucocerebrosidase following retroviral vector-mediated transduction of murine hematopoietic stem cells.

The human glucocerebrosidase (GC) gene has been expressed in the progeny of murine hematopoietic stem cells following transduction of marrow with a retroviral vector (G2) containing the human GC cDNA. Murine marrow was transduced via co-cultivation following prestimulation in the presence or absence of recombinant IL-3 and IL-6. A high rate of gene transfer and expression (95%) was demonstrated in primary day 12 CFU-S foci following bone marrow transplantation (BMT) of G2-transduced marrow into lethally irradiated syngeneic recipient mice. Immunoreactive human GC protein was also documented in the CFU-S foci. Primary recipient mice were examined 4-6 months following BMT. A higher rate of gene transfer (87%) was seen in hematopoietic organs of recipients of prestimulated donor marrow compared with organs from initially unstimulated marrow (25%). A high rate of expression of human GC was also documented in the prestimulated organs (50%) when compared with the unstimulated group (25%). Secondary BMT was performed using marrow from the long-lived primary recipients. The human GC gene was present in 88% of secondary day 12 CFU-S foci examined in the prestimulated group versus 23% in the unstimulated group. Expression of the human GC gene was documented in secondary day 12 CFU-S foci, providing strong evidence of initial hematopoietic stem cell transduction.     

Adapting a transforming growth factor beta-related tumor protection strategy to enhance antitumor immunity

Transforming growth factor beta (TGF-beta), a pleiotropic cytokine that regulates cell growth and differentiation, is secreted by many human tumors and markedly inhibits tumor-specific cellular immunity. Tumors can avoid the differentiating and apoptotic effects of TGF-beta by expressing a nonfunctional TGF-beta receptor. We have determined whether this immune evasion strategy can be manipulated to shield tumor-specific cytotoxic T lymphocytes (CTLs) from the inhibitory effects of tumor-derived TGF-beta. As our model we used Epstein-Barr virus (EBV)-specific CTLs that are infused as treatment for EBV-positive Hodgkin disease but that are vulnerable to the TGF-beta produced by this tumor. CTLs were transduced with a retrovirus vector expressing the dominant-negative TGF-beta type II receptor HATGF-betaRII-Deltacyt. HATGF-betaRII-Deltacyt- but not green fluorescence protein (eGFP)-transduced CTLs was resistant to the antiproliferative and anticytotoxic effects of exogenous TGF-beta. Additionally, receptor-transduced cells continued to secrete cytokines in response to antigenic stimulation. TGF-beta receptor ligation results in phosphorylation of Smad2, and this pathway was disrupted in HATGF-betaRII-Deltacyt-transduced CTLs, confirming blockade of the signal transduction pathway. Long-term expression of TGF-betaRII-Deltacyt did not affect CTL function, phenotype, or growth characteristics. Tumor-specific CTLs expressing HATGF-betaRII-Deltacyt should have a selective functional and survival advantage over unmodified CTLs in the presence of TGF-beta-secreting tumors and may be of value in treatment of these diseases.     

Analysis of gene amplification in human tumor cell lines.

Oncogene amplification has been observed in various primary tumors and tumor-derived cell lines. In several types of cancer, amplification of specific oncogenes is correlated with the stage of tumor progression. To estimate the frequency of gene amplification in other tumor types and to determine whether the ability to grow in vivo is associated with gene amplification in tumor cell lines, we have developed a modified version of the in-gel renaturation assay that detects human DNA sequences of unknown nature amplified as little as 7- to 8-fold. This assay was used to screen 16 cell lines derived from various solid tumors and leukemias. Amplified DNA sequences were detected in only one cell line, Calu-3 lung adenocarcinoma. This cell line was found to contain coamplified NGL (formerly termed neu) and ERBA1 oncogenes. However, when one of the amplification-negative cell lines, PC-3 prostatic carcinoma, was selected for in vivo growth in nude mice, amplified DNA sequences became detectable in these cells. The amplified sequences included the MYC oncogene, which showed no amplification in the parental cell line but was amplified 10- to 12-fold in the in vivo-selected cells. MYC amplification may, therefore, provide tumor cells with a selective advantage specific for in vivo growth.