Expression of cyclins E1 and E2 during mouse development and in neoplasia

Abstract

Cyclin E1 (formerly called cyclin E) and the recently described cyclin E2 belong to the family of E-type cyclins that operate during the G(1)/S phase progression in mammalian cells. The two E-cyclins share a catalytic partner, cyclin-dependent kinase 2 (CDK2), and activate their associated kinase activities at similar times during cell cycle progression. Despite these similarities, it is unknown whether the two proteins perform distinct functions, or, alternatively, they control S-phase entry of different cell types in a tissue-specific fashion. To start addressing in vivo functions of E-cyclins, we determined the expression pattern of cyclins E1 and E2 during normal mouse development. We found that the two E-cyclins showed very similar patterns of expression; both were expressed within the proliferating compartment during embryo development. Analyses of cells and tissues lacking members of the retinoblastoma (pRB) family of proteins revealed that the expression of both cyclins is controlled in a pRB-dependent, but p107- and p130-independent fashion, likely through the pRB-dependent E2F transcription factors. We also found that cyclins E1 and E2 are expressed at high levels in mouse breast tumors driven by the Myc oncogene. Last, we found that cyclin E2 is overexpressed in approximately 24% of analyzed human mammary carcinomas. Collectively these findings suggest that the expression of cyclins E1 and E2 is governed by similar molecular circuitry.

Figure 1

Detection of cyclin E1 and E2 mRNA in developing mouse embryos, by in situ hybridization. Adjacent sections from embryos collected at indicated stages of development were hybridized with probes specific for cyclin E1 or cyclin E2. Red represents the hybridization signal. Blue represents the Hoechst 33258 staining of cell nuclei. The composite images of the whole embryo were obtained by assembling overlapping pictures taken at the same exposure times. (Right) Sections stained for BrdUrd. n, Neuroepithelium; m, mandibular component of first branchial arc; h, hepatic primordium; b, hindlimb bud; lv, liver; mb, midbrain, d, duodenum; lu, lung, i, intestine. (Magnifications: day E9.5, ×22; day E10.5, ×12; day E11.5, ×11; day E13.5, ×11.)

Figure 2

Expression of E-cyclin mRNAs in wild-type mice. (a) RNA was isolated from indicated organs of adult, 2- to 4-month-old, wild-type mice. Northern blots were probed with cyclin E1- and cyclin E2-specific probes. (b) RNA was isolated from wild-type embryos (Emb), mouse embryonal fibroblasts (MEFs), and embryonal stem cells (ES cells). Blots were probed with cyclin E1- and cyclin E2-specific probes. In both a and b, the bottom gels have been stained with ethidium bromide.

Figure 3

Expression of E-cyclin mRNAs in mutant mice. (a) RNA was isolated from brains derived from day E13.5 wild-type (WT) or pRB^−/− embryos. Northern blots were hybridized with cyclin E1- and E2-specific probes. The bottom gel has been stained with ethidium bromide. (b) Wild-type or pRB^−/− mouse embryonal fibroblasts (MEFs) were rendered quiescent by serum deprivation and then stimulated to enter the cell cycle by serum re-addition. Cells were harvested at indicated time points (in hours), and RNA was extracted and processed for Northern blot analyses. Blots were hybridized with probes specific for cyclin E1 or cyclin E2, or with a probe specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. (c) Same type of experiment as in b, except that MEFs were prepared from p130^+/− or p130-deficient or p130/p107-deficient embryos.

Figure 4

Expression of E-cyclins in mouse breast tumors. RNA was isolated from mammary glands of nontransgenic, wild-type mice (WT MG), from breast tumors arising in transgenic MMTV-v-Ha-Ras female mice (MMTV/Ras T), or from breast tumors arising in transgenic MMTV-c-Myc female mice (MMTV/Myc T). Northern blots of representative samples were hybridized with probes specific for cyclin E1 or E2. (Bottom) Ethidium bromide-stained gel.

Figure 5

Expression of E-cyclins in human breast cancers. (a) Examples of Northern blots of human breast cancer samples and normal mammary glands (normal) hybridized with cyclin E2-specific probe. The symbols of tumor samples are indicated above the respective lanes. Cyclin E2-overexpressing samples are marked with stars. RNA isolated from an immortalized human mammary epithelial cell line (HME) served as a positive control. (b) Selected tumor samples were rerun, and Northern blots were probed with cyclin E1- and E2-specific probes. RNA isolated from normal mammary glands (normal) and from the HME cell line was also included as in a. Cyclin E1-overexpressing samples are marked with stars. The bottom gels have been stained with ethidium bromide.