Interaction of mouse polycomb-group (Pc-G) proteins Enx1 and Enx2 with Eed: indication for separate Pc-G complexes.

The Polycomb group (Pc-G) constitutes an important, functionally conserved group of proteins, required to stably maintain inactive homeobox genes repressed during development. Drosophila extra sex combs (esc) and its mammalian homolog embryonic ectoderm development (eed) are special Pc-G members, in that they are required early during development when Pc-G repression is initiated, a process that is still poorly understood. To get insight in the molecular function of Eed, we searched for Eed-interacting proteins, using the yeast two-hybrid method. Here we describe the specific in vivo binding of Eed to Enx1 and Enx2, two mammalian homologs of the essential Drosophila Pc-G gene Enhancer-of-zeste [E(z)]. No direct biochemical interactions were found between Eed/Enx and a previously characterized mouse Pc-G protein complex, containing several mouse Pc-G proteins including mouse polyhomeotic (Mph1). This suggests that different Pc-G complexes with distinct functions may exist. However, partial colocalization of Enx1 and Mph1 to subnuclear domains may point to more transient interactions between these complexes, in support of a bridging role for Enx1.

CDK-independent activation of estrogen receptor by cyclin D1.

Both cyclin D1 and estrogens have an essential role in regulating proliferation of breast epithelial cells. We show here a novel role for cyclin D1 in growth regulation of estrogen-responsive tissues by potentiating transcription of estrogen receptor-regulated genes. Cyclin D1 mediates this activation independent of complex formation to a CDK partner. Cyclin D1 activates estrogen receptor-mediated transcription in the absence of estrogen and enhances transcription in its presence. The activation of estrogen receptor by cyclin D1 is not inhibited by anti-estrogens. A direct physical binding of cyclin D1 to the hormone binding domain of the estrogen receptor results in an increased binding of the receptor to estrogen response element sequences, and upregulates estrogen receptor-mediated transcription. These results highlight a novel role for cyclin D1 as a CDK-independent activator of the estrogen receptor.

Cyclin D1 triggers autonomous growth of breast cancer cells by governing cell cycle exit.

Cyclin D1 controls G1-associated processes, including G0-to-G1 and G1-to-S transitions. This study demonstrates a novel aspect of cyclin D1 as a regulator of the transition between G1 and G0. Overexpression of cyclin D1 in MCF7 breast tumor cells resulted in a continued proliferation under low-serum conditions, whereas nonoverexpressing cells ceased to grow. This difference in growth was due to a reduced exit from G1 to G0 in cyclin D1-overexpressing cells. Our data therefore suggest a model in which cyclin D1 overexpression in tumor cells is responsible for hyperproliferation under growth factor-deprived conditions.

A clinicopathological study on overexpression of cyclin D1 and of p53 in a series of 248 patients with operable breast cancer.

Overexpression of cyclin D1 is frequently found in various types of human tumours and results from clonal rearrangement and/or amplification involving chromosomal region 11q13. In order to evaluate the pathological relevance of cyclin D1 overexpression in human breast cancer, we generated a polyclonal antiserum against the carboxy-terminal part of the cyclin D1 protein. After affinity purification, the antiserum specifically detected overexpression of cyclin D1 in formalin-fixed, paraffin-embedded tumour material also. The intensity of the nuclear stainings was, in general, proportional to the degree of cyclin D1 amplification. We did not encounter significant variability of staining within individual tumours with overexpression of cyclin D1. Overexpression of cyclin D1 appeared to be associated with oestrogen receptor-positive breast tumours, but not with any other clinicopathological parameter tested. Overexpression of cyclin D1 was not prognostic value for recurrence of survival in a consecutive series of 248 operable breast cancer patients (stage I and II). Overexpression of p53 was also not of prognostic significance in this series, but was associated with undifferentiated histology and oestrogen receptor-negative breast tumours, as has been reported previously by others. A high proportion of breast tumours with a low grade of malignancy in this series of operable breast cancer patients may explain discrepancies concerning the prognostic value of amplification and of overexpression of cyclin D1.

Overexpression of cyclin D1 correlates with recurrence in a group of forty-seven operable squamous cell carcinomas of the head and neck.

We evaluated the prognostic significance of overexpression of cyclin D1 in 47 patients with surgically resected squamous cell carcinomas of the head and neck. Overexpression of cyclin D1 was detected immunohistochemically using an affinity-purified polyclonal antibody directed against the carboxyl-terminal part of the cyclin D1 protein, applied to formalin-fixed, paraffin-embedded tissue sections. Overexpression of cyclin D1 was found in 30 of 47 head and neck squamous cell carcinoma (HNSCC) cases and was associated with a more rapid and frequent recurrence of disease (P = 0.027). There was a 5-year disease-free interval of 47% for HNSCC patients with a strong overexpression of cyclin D1 and of 80% for cyclin D1-negative HNSCC patients. Overexpression of cyclin D1 was also associated with a shortened overall survival of these patients (P = 0.0095), with a 5-year survival of 60% for the cyclin D1 strongly positive cases and of 83% for cyclin D1-negative cases. Overexpression of cyclin D1 appears to indicate poor prognosis in operable HNSCC.

Sequence similarity between the mammalian bmi-1 proto-oncogene and the Drosophila regulatory genes Psc and Su(z)2.

The bmi-1 proto-oncogene can be activated by Moloney murine leukaemia proviral insertions in E mu-myc transgenic mice. It encodes a highly conserved nuclear protein of 324 amino acids which belongs to a family of proteins containing a putative new zinc-finger. Another closely related member of this family is the mouse protein Mel-18. Here we report on the cloning and characterization of a homologous gene (D-bmi) from Drosophila melanogaster. Our analysis indicates that distinct domains of the mouse Bmi-1 protein, including the putative zinc-finger motif, are highly conserved within the much larger D-Bmi protein. Chromosomal localization and sequence comparison reveal that D-bmi is identical to Posterior Sex Combs (Psc) and indicate that the conserved domains between mouse bmi and Psc are also conserved within Suppressor-2 of Zeste (Su(z)2).

Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging.

Mo-MLV infection of E mu-myc transgenic mice results in a dramatic acceleration of pre-B cell lymphomagenesis. We have used provirus tagging to identify genes that cooperate with the E mu-myc transgene in B cell transformation. Here we report on the identification of four loci, pim-1, bmi-1, pal-1, and bla-1, which are occupied by proviruses in 35%, 35%, 28%, and 14% of the tumors, respectively. bmi-1, pal-1, and bla-1 represent novel common proviral insertion sites. The bmi-1 gene encodes a 324 amino acid protein with a predominantly nuclear localization. bmi-1 is highly conserved in evolution and contains several motifs frequently found in transcriptional regulators, including a new putative zinc finger motif. No genes have yet been assigned to pal-1 and bla-1. The distribution of proviruses over the four common insertion sites suggests that provirus tagging can be used not only to identify the cooperating oncogenes but also to assign these genes to distinct complementation groups in tumorigenesis.

Carcinogen-induced lymphomagenesis in pim-1 transgenic mice: dose dependence and involvement of myc and ras.

Transgenic mice overexpressing the pim-1 oncogene in their lymphoid compartments are predisposed to T-cell lymphomagenesis but only to the extent that approximately 10% of the transgenic mice develop lymphomas within 34 weeks after birth. Recently, we have shown that lymphomagenesis in pim-1 transgenic mice can be accelerated by infecting pim-1 transgenic mice with murine leukemia viruses or by treating the mice with a relatively low dose of 60 mg of the carcinogen N-ethyl-N-nitrosourea (ENU) per kg of body weight. Here we describe the incidence of tumors as a function of the dose of ENU. Either 200, 15, 4, 1, or 0.1 mg/kg ENU was injected into transgenic and control mice and the tumor incidence was monitored. T-cell lymphomas developed in 100 and 70% of the pim-1 transgenic mice treated with 200 and 15 mg/kg ENU, respectively. Approximately 20% of the Emu-pim-1 transgenic mice developed lymphomas after treatment with either 4, 1, or 0.1 mg/kg ENU. The nontransgenic mice developed lymphomas only after injection with 200 mg/kg (45%). The data show that Emu-pim-1 transgenic mice are approximately 25-fold more susceptible to ENU-induced lymphomagenesis than control mice. In most tumors the expression of c-myc was strongly elevated, probably as a direct or indirect effect of ENU. Analysis of the lymphomas for ras mutations revealed that approximately 10% of the lymphomas bear a ras mutation. The data indicate that at least some of these mutations are not the direct result of alkylation by ENU but rather represent spontaneous mutations that occurred later in the tumorigenic process.

Very high frequency of lymphoma induction by a chemical carcinogen in pim-1 transgenic mice.

Infection of mice with Moloney murine leukaemia virus (MuLV) induces T-cell lymphomas after an average latency period of 150 days. In these lymphomas the MuLV DNA is frequently integrated into the mouse chromosomal DNA in the vicinity of the pim-1 oncogene. Transgenic mice overexpressing the pim-1 oncogene are predisposed to develop T-cell lymphomas, but only to the extent that approximately 10% of the mice develop a lymphoma within 240 days. When these mice are infected with MuLV, lymphomas develop in all mice in only 50-60 days. In these lymphomas MuLV DNA is integrated near either the c-myc or N-myc gene, suggesting that pim-1 and myc synergize in lymphomagenesis. To determine whether this system has a more general application, we have now tested the susceptibility of pim-1 transgenic mice to N-ethyl-N-nitrosourea (ENU), a chemical carcinogen. With a single low dose of ENU, nearly all pim-1 transgenic mice, but only 15% of non-transgenic mice, develop T-cell lymphomas within 200 days. All ENU-induced lymphomas in both pim-1 transgenic and non-transgenic mice express high levels of c-myc messenger RNA, supporting the notion that pim-1 and c-myc synergize in lymphoma induction. We propose that pim-1 transgenic mice could be used to test the oncogenic potential of other chemical compounds.