Ki-67 as a molecular target for therapy in an in vitro three-dimensional model for ovarian cancer.

Targeting molecular markers and pathways implicated in cancer cell growth is a promising avenue for developing effective therapies. Although the Ki-67 protein (pKi-67) is a key marker associated with aggressively proliferating cancer cells and poor prognosis, its full potential as a therapeutic target has never before been successfully shown. In this regard, its nuclear localization presents a major hurdle because of the need for intracellular and intranuclear delivery of targeting and therapeutic moieties. Using a liposomally encapsulated construct, we show for the first time the specific delivery of a Ki-67-directed antibody and subsequent light-triggered death in the human ovarian cancer cell line OVCAR-5. Photoimmunoconjugate-encapsulating liposomes (PICEL) were constructed from anti-pKi-67 antibodies conjugated to fluorescein 5(6)-isothiocyanate, as a photoactivatable agent, followed by encapsulation in noncationic liposomes. Nucleolar localization of the PICELs was confirmed by confocal imaging. Photodynamic activation with PICELs specifically killed pKi-67-positive cancer cells both in monolayer and in three-dimensional (3D) cultures of OVCAR-5 cells, with the antibody TuBB-9 targeting a physiologically active form of pKi-67 but not with MIB-1, directed to a different epitope. This is the first demonstration of (a) the exploitation of Ki-67 as a molecular target for therapy and (b) specific delivery of an antibody to the nucleolus in monolayer cancer cells and in an in vitro 3D model system. In view of the ubiquity of pKi-67 in proliferating cells in cancer and the specificity of targeting in 3D multicellular acini, these findings are promising and the approach merits further investigation.

HP1gamma function is required for male germ cell survival and spermatogenesis.

BACKGROUND: HP1 proteins are conserved components of eukaryotic constitutive heterochromatin. In mammals, there are three genes that encode HP1-like proteins, termed HP1alpha, HP1beta and HP1gamma, which have a high degree of homology This paper describes for the first time, to our knowledge, the physiological function of HP1gamma using a gene-targeted mouse. RESULTS: While targeting the Cbx3 gene (encoding the HP1gamma protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1gamma protein. Homozygotes for the hypomorphic allele (Cbx3hypo/hypo) are rare, with only 1% of Cbx3hypo/hypo animals reaching adulthood. Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype). The percentage of seminiferous tubules that are positive for L1 ORF1 protein (ORF1p) in Cbx3hypo/hypo testes is greater than that for wild-type testes, indicating that L1 retrotransposon silencing is reversed, leading to ectopic expression of ORF1p in Cbx3hypo/hypo germ cells. CONCLUSIONS: The Cbx3 gene product (the HP1gamma protein) has a non-redundant function during spermatogenesis that cannot be compensated for by the other two HP1 isotypes. The Cbx3hypo/hypo spermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants. The Cbx3 gene-targeted mice generated in this study provide an appropriate model for the study of HP1gamma in transposon silencing and parental imprinting.

Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in beta-thalassemia.

In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

Plasticity of HP1 proteins in mammalian cells.

We have compared the distribution of endogenous heterochromatin protein 1 (HP1) proteins (alpha, beta and gamma) in different epithelial lines, pluripotent stem cells and embryonic fibroblasts. In parallel, we have interrogated assembly and dynamics of newly expressed HP1-GFP proteins in cells lacking both HP1alpha and HP1beta alleles, blocked at the G1-S boundary, or cultured in the presence of HDAC and HAT inhibitors. The results reveal a range of cell type and differentiation state-specific patterns that do not correlate with 'fast' or 'slow' subunit exchange in heterochromatin. Furthermore, our observations show that targeting of HP1gamma to heterochromatic sites depends on HP1alpha and H1beta and that, on an architectural level, HP1alpha is the most polymorphic variant of the HP1 family. These data provide evidence for HP1 plasticity under shifting microenvironmental conditions and offer a new conceptual framework for understanding chromatin dynamics at the molecular level.

Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells.

The nuclear Ki-67 protein (pKi-67) has previously been shown to be exclusively expressed in proliferating cells. As a result, antibodies against this protein are widely used as prognostic tools in cancer diagnostics. Here we show, that despite the strong downregulation of pKi-67 expression in non-proliferating cells, the protein can nevertheless be detected at sites linked to ribosomal RNA (rRNA) synthesis. Although this finding does not argue against the use of pKi-67 as a proliferation marker, it has wide ranging implications for the elucidation of pKi-67 function. Employing the novel antibody TuBB-9, we could further demonstrate that also in proliferating cells, a fraction of pKi-67 is found at sites linked to the rRNA transcription machinery during interphase and mitosis. Moreover, chromatin immunoprecipitation (ChIP) assays provided evidence for a physical association of pKi-67 with chromatin of the promoter and transcribed region of the rRNA gene cluster. These data strongly suggest a role for pKi-67 in the early steps of rRNA synthesis.