IDENTIFICATION AND CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES IN TUMOR METASTASIS:
THE nm23 GENE
Patricia S. Steeg, Generoso Bevilacqua, Mark E. Sobel, and Lance A. Liotta Laboratory of Pathology National Cancer Institute National Institutes of Health Bethesda, MD 20892 Tumor metastasis is a complex process involving tumor cell invasion, locomotion, intravasation and extravasation of the circulatory system, angiogenesis, colony formation, and avoidance of host immunological responses. Two premises have guided our investigation into the genetic influences on tumor invasion and metastasis. First, if the metastatic process is regulated, at least in part, by the activation and deactivation of specific genes, then the multiplicity of cell functions in metastasis dictates that many genes are involved. Second, the biochemical nature of molecules regulating and executing each of the tumor cell functions in metastasis is incompletely understood. Because of the tedious purification process, it is likely that many metastasis regulatory and effector compounds and the genes encoding them are presently unknown. Based on these premises, we initiated differential colony hybridization experiments to identify genes associated with the tumor invasion and metastatic process. This technique identifies genes either activated or deactivated between tumor cells of low and high metastatic potential. It can therefore identify metastasis-related genes in advance of conventional biochemical purification and DNA cloning. This paper describes the identification and characterization of one such gene, nm23. IDENTIFICATION OF THE nm23 GENE We utilized seven cell lines derived from a single K-1735 murine melanoma (Fidler et al., 1981; Fidler, 1984; Kalebic et al., 1988) to identify genes differentially expressed in tumor metastasis. These cell lines exhibit significant differences in spontaneous and experimental metastatic potential (Steeg et al., 1988). In vitro translations of total cellular RNA from each K-1735 cell line are shown in Fig. 1. Most of the bands are uniformly expressed between the low metastatic potential clones 16 and 19 and the high metastatic potential M2, M4, Tk, Tk-Eve and Tk-liver lines, confirming their similarity. However, several bands were differentially expressed between the low and high metastatic potential cell lines. These differentially expressed bands indicate that specific rnRNAs are differentially regulated in the K-1735 system. A 40,000 component cDNA library was constructed by G/C tailing K-1735 cDNAs into the PstI site of pBR322. Duplicate filters of the library were Boundaries between Promotion and Progression during Carcinogenesis Edited by O. Sudilovsky et al., Plenum Press, New York, 1991
355
hybridized to 32P-labeled mRNAs from a low metastic (clone 19) and a high metastatic (Tk-Eve) K-1735 cell line. Of 24 cDNA clones identified that were differentially expressed between these two K-1735 cell lines, the biology of one clone, pnm23, has been extremely interesting. Expression of the nm23 gene has been consistently down-regulated in tumor cells of high metastatic potential in four experimental models of tumor metastasis. K-1735 MURINE MELANOMAS The nm23 RNA levels of seven K-1735 melanoma cell lines were determined by Northern blot hybridization (Steeg et al., 1988). nm23 RNA levels in the low metastatic K-1735 clone 16 and clone 19 cell lines are 10-fold higher than in five related, high metastatic cell lines (Fig. 2). In situ hybridization experiments indicate that virtually all tumor cells express high nm23 RNA levels, as opposed to a subpopulation of cells (Steeg et al., 1988).----
kDa -NM
-MAP
200-
-NM
97.4-
-MAP -MAP
68-
43-MAP CI16
CI19
M2
M4
TK
TK·Eve TK·Liver
Fig. 1. In vitro translation of K-1735 melanoma RNAs. One microgram of total cellular RNA from each K-1735 cell line was translated in vitro using 3sS-methionine and a rabbit reticulocyte lysate, and the translation products electrophoresed on a 7% SDS-PAGE gel, a fluorograph of which is shown. Indicated by MAP are metastases associated proteins, bands expressed to a greater degree by the high metastatic potential M2, M4, TK, TK-Eve and TK-liver lines than the low metastatic clones 16 and 19 lines. Conversely, nm denotes bands expressed to a greater degree by the low metastatic K-1735 lines. 356
ONCOGENE-TRANSFECTED RAT EMBRYO FIBROBLASTS To confirm that the prum23 cDNA clone was associated with the tumor metastatic process, rum23 RNA levels were determined in additional metastasic experimental systems. rum23 RNA levels in three low metastatic, c-Ha-ras + adenovirus 2 E1a cotransfected rat embryo fibroblast (REF) lines were 2- to 8-fold higher~an in three control, high metastatic c-Ha-ras-transfected REF lines (Steeg et al., 1989). Again, in situ hybridization experiments indicated that the high rum23 RNA levels observed in the low metastatic REF
K1735 Cell Une : Median Experimental Pulmonary Metastases : Range:
c:: 0
~
01>
> w
01>
c::
u u 0
6
0
O· 10
O·
1
THE nm23 GENE
Patricia S. Steeg, Generoso Bevilacqua, Mark E. Sobel, and Lance A. Liotta Laboratory of Pathology National Cancer Institute National Institutes of Health Bethesda, MD 20892 Tumor metastasis is a complex process involving tumor cell invasion, locomotion, intravasation and extravasation of the circulatory system, angiogenesis, colony formation, and avoidance of host immunological responses. Two premises have guided our investigation into the genetic influences on tumor invasion and metastasis. First, if the metastatic process is regulated, at least in part, by the activation and deactivation of specific genes, then the multiplicity of cell functions in metastasis dictates that many genes are involved. Second, the biochemical nature of molecules regulating and executing each of the tumor cell functions in metastasis is incompletely understood. Because of the tedious purification process, it is likely that many metastasis regulatory and effector compounds and the genes encoding them are presently unknown. Based on these premises, we initiated differential colony hybridization experiments to identify genes associated with the tumor invasion and metastatic process. This technique identifies genes either activated or deactivated between tumor cells of low and high metastatic potential. It can therefore identify metastasis-related genes in advance of conventional biochemical purification and DNA cloning. This paper describes the identification and characterization of one such gene, nm23. IDENTIFICATION OF THE nm23 GENE We utilized seven cell lines derived from a single K-1735 murine melanoma (Fidler et al., 1981; Fidler, 1984; Kalebic et al., 1988) to identify genes differentially expressed in tumor metastasis. These cell lines exhibit significant differences in spontaneous and experimental metastatic potential (Steeg et al., 1988). In vitro translations of total cellular RNA from each K-1735 cell line are shown in Fig. 1. Most of the bands are uniformly expressed between the low metastatic potential clones 16 and 19 and the high metastatic potential M2, M4, Tk, Tk-Eve and Tk-liver lines, confirming their similarity. However, several bands were differentially expressed between the low and high metastatic potential cell lines. These differentially expressed bands indicate that specific rnRNAs are differentially regulated in the K-1735 system. A 40,000 component cDNA library was constructed by G/C tailing K-1735 cDNAs into the PstI site of pBR322. Duplicate filters of the library were Boundaries between Promotion and Progression during Carcinogenesis Edited by O. Sudilovsky et al., Plenum Press, New York, 1991
355
hybridized to 32P-labeled mRNAs from a low metastic (clone 19) and a high metastatic (Tk-Eve) K-1735 cell line. Of 24 cDNA clones identified that were differentially expressed between these two K-1735 cell lines, the biology of one clone, pnm23, has been extremely interesting. Expression of the nm23 gene has been consistently down-regulated in tumor cells of high metastatic potential in four experimental models of tumor metastasis. K-1735 MURINE MELANOMAS The nm23 RNA levels of seven K-1735 melanoma cell lines were determined by Northern blot hybridization (Steeg et al., 1988). nm23 RNA levels in the low metastatic K-1735 clone 16 and clone 19 cell lines are 10-fold higher than in five related, high metastatic cell lines (Fig. 2). In situ hybridization experiments indicate that virtually all tumor cells express high nm23 RNA levels, as opposed to a subpopulation of cells (Steeg et al., 1988).----
kDa -NM
-MAP
200-
-NM
97.4-
-MAP -MAP
68-
43-MAP CI16
CI19
M2
M4
TK
TK·Eve TK·Liver
Fig. 1. In vitro translation of K-1735 melanoma RNAs. One microgram of total cellular RNA from each K-1735 cell line was translated in vitro using 3sS-methionine and a rabbit reticulocyte lysate, and the translation products electrophoresed on a 7% SDS-PAGE gel, a fluorograph of which is shown. Indicated by MAP are metastases associated proteins, bands expressed to a greater degree by the high metastatic potential M2, M4, TK, TK-Eve and TK-liver lines than the low metastatic clones 16 and 19 lines. Conversely, nm denotes bands expressed to a greater degree by the low metastatic K-1735 lines. 356
ONCOGENE-TRANSFECTED RAT EMBRYO FIBROBLASTS To confirm that the prum23 cDNA clone was associated with the tumor metastatic process, rum23 RNA levels were determined in additional metastasic experimental systems. rum23 RNA levels in three low metastatic, c-Ha-ras + adenovirus 2 E1a cotransfected rat embryo fibroblast (REF) lines were 2- to 8-fold higher~an in three control, high metastatic c-Ha-ras-transfected REF lines (Steeg et al., 1989). Again, in situ hybridization experiments indicated that the high rum23 RNA levels observed in the low metastatic REF
K1735 Cell Une : Median Experimental Pulmonary Metastases : Range:
c:: 0
~
01>
> w
01>
c::
u u 0
6
0
O· 10
O·
1
