Blasi E, Radzioch D, Merletti L, Varesio L
Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick Cancer Research Facility, Maryland 21701.
Cancer Biochem Biophys. 1989 Oct;10(4):303-17.
We have studied the effects of infection of fresh murine bone marrow (BM) cells by recombinant retroviruses carrying v-raf and v-myc oncogenes, either alone or in combination. Viruses containing v-raf or v-myc alone failed to induce BM proliferation in 24 out of 27 experiments performed so far, only the J2 virus containing both v-raf and v-myc oncogenes induced BM proliferation. Exogenous growth factors (GF) were not required to sustain the mitogenic effect of J2 virus. Infection with retroviruses carrying only v-raf or v-myc did not induce BM cell growth, indicating that co-expression of the two oncogenes was needed to provide the mitogenic signal(s) for BM proliferation. The kinetics of growth of the J2 virus-infected cells (J2 cells) were characteristically biphasic. The initial burst of proliferation was always followed by a quiescent phase culminating in cell death, which could not be reversed by addition of exogenous GF. In contrast, active proliferation of the quiescent monolayers could be restored by addition of dextran-based beads to the cultures, showing that the growth arrest of J2 cells was a reversible process. J2 cells actively growing in the presence of CT-beads could be expanded and cloned and subsequently grew continuously independent of the CT-beads. Eighteen clones obtained from different infections were all macrophages (M phi) by morphological criteria and all of them expressed the same membrane phenotype compatible with M phi, demonstrating that J2 virus infection leads to immortalization of the same BM-derived monocytic subpopulation. When injected in vivo, J2 cells produced histiocytic tumors in nude mice, but did not grow in immunocompetent syngeneic mice. The cells induced to proliferate in vitro in response to J2 virus infection appeared to be limited to the BM compartment, since spleen cells, thymocytes, peritoneal M phi and liver large granular lymphocytes did not grow in vitro in response to J2 virus. The immortalization of BM cells by J2 virus infection represents a novel reproducible experimental system to deliberately generate M phi lines, which proliferate in response to viral oncogenes and do not require exogenous GF to initiate or to sustain their continuous proliferation.
我们研究了携带v-raf和v-myc癌基因的重组逆转录病毒单独或联合感染新鲜小鼠骨髓（BM）细胞的效果。到目前为止，在27次实验中的24次实验里，仅携带v-raf或v-myc的病毒未能诱导BM增殖，只有同时包含v-raf和v-myc癌基因的J2病毒能诱导BM增殖。维持J2病毒的促有丝分裂作用无需外源性生长因子（GF）。仅携带v-raf或v-myc的逆转录病毒感染不会诱导BM细胞生长，这表明这两个癌基因的共表达是为BM增殖提供促有丝分裂信号所必需的。J2病毒感染细胞（J2细胞）的生长动力学具有典型的双相性。最初的增殖爆发之后总是接着一个静止期，最终导致细胞死亡，添加外源性GF无法逆转这种情况。相反，向培养物中添加基于葡聚糖的珠子可以恢复静止单层细胞的活跃增殖，这表明J2细胞的生长停滞是一个可逆过程。在CT-珠子存在下活跃生长的J2细胞可以扩增并克隆，随后独立于CT-珠子持续生长。根据形态学标准，从不同感染中获得的18个克隆均为巨噬细胞（M phi），并且它们都表达与M phi相容的相同膜表型，这表明J2病毒感染导致相同的BM来源单核细胞亚群永生化。当注射到体内时，J2细胞在裸鼠中产生组织细胞瘤，但在具有免疫活性的同基因小鼠中不生长。由于脾细胞、胸腺细胞、腹膜M phi和肝大颗粒淋巴细胞不会因J2病毒而在体外生长，因此在体外响应J2病毒感染而被诱导增殖的细胞似乎仅限于BM区室。J2病毒感染使BM细胞永生化代表了一种新型的可重复实验系统，可特意产生M phi系，这些细胞响应病毒癌基因而增殖，并且启动或维持其持续增殖不需要外源性GF。
