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.

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.

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.

Tissue microarrays from HOPE-fixed specimens allow for enhanced high throughput molecular analyses in paraffin-embedded material.

In this study, we describe a technique that allows for the production and the use of tissue microarrays (TMA) from HOPE-fixed, paraffin-embedded specimens. The combination of these two technologies unites the advantages of the high throughput aspects in TMA, with good preservation of nucleic acids, proteins, and morphology of HOPE-fixed tissues, thus substantially widening the capabilities presently available for molecular studies in paraffin-embedded tissues.

Topographic and quantitative display of integrated human immunodeficiency virus-1 provirus DNA in human lymph nodes by real-time polymerase chain reaction.

In situ polymerase chain reaction (isPCR) has been applied in many fields that require detection of a genomic marker in combination with its topographic localization in tissue. We describe here a novel approach that circumvents the major drawbacks of in situ PCR, ie, low sensitivity, leakage of DNA from cells, and inability to quantify the DNA input. Frozen sections of a lymph node from a human immunodeficiency virus (HIV)-1-infected patient were fixed on glass microscope slides, and the glass was scored into square fragments of 0.5-mm edge length using a diamond cutting device. Slides were then attached to adhesive, elastic plastic foil and finally broken, and the foil was extended to allow sorting of fragments into PCR microtiter plates. The material was tested for HIV-1 proviral DNA by a sensitive real-time PCR protocol. Subjacent sections were stained for follicular dendritic cells to identify follicles. The fragmentation process prevented leakage of amplified DNA to neighboring areas as often experienced with in situ PCR. Provirus was clearly associated with follicular areas, in which provirus-carrying cells represented an average of 0.8% of the total cell population (peak density, 3.1% of all follicular cells). The results of this method suggest that the high density of provirus-containing cells in follicles may be important for the persistence of proviral DNA in infected persons.

Human lung cancer cells express functionally active Toll-like receptor 9.

BACKGROUND: CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology. METHODS: The expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system. RESULTS: We found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis. CONCLUSIONS: Here we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology.

Elevation of plasma membrane permeability by laser irradiation of selectively bound nanoparticles.

Irradiation of nanoabsorbers with pico- and nanosecond laser pulses could result in thermal effects with a spatial confinement of less than 50 nm. Therefore absorbing nanoparticles could be used to create controlled cellular effects. We describe a combination of laser irradiation with nanoparticles, which changes the plasma membrane permeability. We demonstrate that the system enables molecules to penetrate impermeable cell membranes. Laser light at 532 nm is used to irradiate conjugates of colloidal gold, which are delivered by antibodies to the plasma membrane of the Hodgkin's disease cell line L428 and/or the human large-cell anaplastic lymphoma cell line Karpas 299. After irradiation, membrane permeability is evaluated by fluorescence microscopy and flow cytometry using propidium iodide (PI) and fluorescein isothiocyanate (FITC) dextran. The fraction of transiently permeabilized and then resealed cells is affected by the laser parameter, the gold concentration, and the membrane protein of the different cell lines to which the nanoparticles are bound. Furthermore, a dependence on particle size is found for these interactions in the different cell lines. The results suggest that after optimization, this method could be used for gene transfection and gene therapy.

Generation and characterization of multicellular heterospheroids formed by human peripheral blood mononuclear cells.

A three-dimensional culture system for primary human mononuclear cells was developed, which reproducibly resulted in the formation of multicellular heterospheroids. Immunohistological characterization demonstrated not only the three-dimensional tissue-like aggregation of primary monocytes, B cells and T cells, but also the presence of macrophages and proliferating cells, indicating that a differentiation of monocytes to macrophages and an activation of cells were induced. Because of the phenotypical resemblance to granulomas the influence of an in vitro infection with mycobacteria on spheroid formation and morphology was analyzed. In comparison to control incubations, the formation of multinucleated giant cells and necrotic areas containing large numbers of mycobacteria could be observed, which resembled histological hallmarks of in situ tuberculoid granulomas. These characteristics have not been described for in vitro models of granuloma formation before and thus this new culture technique may prove to be a useful tool for analyzing aspects relevant to immunopathological processes in chronically inflamed tissues.

Antisense treatment against Ki-67 mRNA inhibits proliferation and tumor growth in vitro and in vivo.

The Ki-67 protein is tightly regulated and depends on the proliferative status of a cell. It is present in the nuclei of proliferating cells but absent in resting cells. Since transformation of malignant cells is frequently associated with high cell proliferation and since proliferation is tightly associated with the Ki-67 protein labeling index, this antigen may represent a potential target for cancer therapy. In the present study we determined the ability of a phosphorothioate antisense oligodeoxyribonucleotide (ODN) targeted against Ki-67 mRNA to inhibit tumor cell proliferation specifically in cell culture, in multicellular 3-dimensional spheroids (MCS) and in subcutaneous murine tumor models. Antisense treatment of 1 myeloid and different epithelial tumor cell lines in suspension and monolayer culture, respectively, resulted in specific reduction of Ki-67 mRNA and protein, inhibition of proliferation and increased apoptotic cell death. Multicellular human bladder carcinoma spheroids lost their 3-dimensional structure and underwent cell death after incubation with antisense oligonucleotides. The growth of subcutaneous syngeneic prostatic (p = 0.05) and transitional cell tumors (p = 0.001) in immunocompetent mice was significantly inhibited in antisense-treated animals. From these findings we conclude that antisense inhibition of Ki-67 protein expression may be a rational approach in anticancer therapy.

Cytoplasmic bacterial lipopolysaccharide does not induce NFkappaB activation or NFkappaB mediated activation signals in human macrophages and an LPS reporter cell line.

Although many membrane components have been described to be involved in the activation of cells by bacterial lipopolysaccharide (LPS), the question remains whether LPS, once internalized by target cells, is also capable of interacting with cytoplasmic elements in such a way that activation of cells results independently of receptor engagement. This is an important aspect to consider with respect to the development of strategies aimed at attenuating adverse effects of LPS in the framework of bacterial infections. In this study, human monocyte derived macrophages as representatives of one of the primary target cells activated by LPS, were microinjected with LPS to circumvent exogenous LPS stimulation. Parameters correlating to cytoplasmic activation of the nuclear transcription factor NFkappaB (intracellular calcium mobilization), to nuclear translocation of the NFkappaB p65 subunit and to mRNA-transcription of inflammatory cytokines known to be expressed upon exogenous LPS-stimulation and to require NFkappaB activation (interleukin-1beta, interleukin-6, tumor necrosis factor alpha) were investigated. In addition, the LPS-reporter cell line 3E10, which contains a reporter gene under the control of an NFkappaB-inducible promoter was analyzed with respect to NFkappaB nuclear translocation and reporter gene expression. None of the cellular systems used and none of the parameters investigated led to the observation that intracellular LPS leads to activation of the cells in comparison to external LPS stimulation. These experiments allow the conclusion that LPS in the cytoplasmic compartment does not lead to NFkappaB translocation, cytokine mRNA transcription, and NFkappaB dependent protein expression and suggest that these activation parameters require the interaction of LPS with external membrane components.

The temporal and spatial distribution of the proliferation associated Ki-67 protein during female and male meiosis.

We used immunolocalization in tissue sections and cytogenetic preparations of female and male gonads to study the distribution of the proliferation marker pKi-67 during meiotic cell cycles of the house mouse, Mus musculus. During male meiosis, pKi-67 was continuously present in nuclei of all stages from the spermatogonium through spermatocytes I and II up to the earliest spermatid stage (early round spermatids) when it appeared to fade out. It was not detected in later spermatid stages or sperm. During female meiosis, pKi-67 was present in prophase I oocytes of fetal ovaries. It was absent in oocytes from newborn mice and most oocytes of primordial follicles from adults. The Ki-67 protein reappeared in oocytes of growing follicles and was continuously present up to metaphase II. Thus, pKi-67 was present in all stages of cell growth and cell division while it was absent from resting oocytes and during the main stages of spermiocytogenesis. Progression through the meiotic cell cycle was associated with extensive intranuclear relocation of pKi-67. In the zygotene and pachytene stages, most of the pKi-67 colocalized with centromeric (centric and pericentric) heterochromatin and adjacent nucleoli; the heterochromatic XY body in male pachytene, however, was free of pKi-67. At early diplotene, pKi-67 was mainly associated with nucleoli. At late diplotene, diakinesis, metaphase I and metaphase II of meiosis, pKi-67 preferentially bound to the perichromosomal layer and was almost absent from the heterochromatic centromeric regions of the chromosomes. After the second division of male meiosis, the protein reappeared at the centromeric heterochromatin and an adjacent region in the earliest spermatid stage and then faded out. The general patterns of pKi-67 distribution were comparable to those in mitotic cell cycles. With respect to the timing, it is interesting to note that relocation from the nucleolus to the perichromosomal layer takes place at the G2/M-phase transition in the mitotic cell cycle but at late diplotene of prophase I in meiosis, suggesting physiological similarity of these stages.

The Ki-67 protein interacts with members of the heterochromatin protein 1 (HP1) family: a potential role in the regulation of higher-order chromatin structure.

The expression of the nuclear protein Ki-67 (pKi-67) is strictly correlated with cell proliferation. Because of this, anti-Ki-67 antibodies can be used as operational markers to estimate the growth fraction of human neoplasia in situ. For a variety of tumours, the assessment of pKi-67 expression has repeatedly been proven to be of prognostic value for survival and tumour recurrence, but no cellular function has yet been ascribed to the Ki-67 protein. This study shows that a C-terminal domain of pKi-67 (Kon21) is able to bind to all three members of the mammalian heterochromatin protein 1 (HP1) family in vitro and in vivo. This interaction can be manipulated in living cells, as evidenced by ectopic expression of GFP-tagged HP1 proteins in HeLa cells, which results in a dramatic relocalization of endogenous pKi-67. Taken together, the data presented in this study suggest a role for pKi-67 in the control of higher-order chromatin structure.

Genotyping in the MHC locus: potential for defining predictive markers in sarcoidosis.

In sarcoidosis, host genetic factors are discussed as contributing to disease susceptibility and course. Since tumor necrosis factor (TNF)-alpha is a central mediator of granuloma formation and since elevated TNF-alpha levels are found during active phases of sarcoidosis, genetic polymorphisms correlating with influences on TNF-alpha levels are of special interest. The complete sequencing of the MHC region and the increase in the number of identified gene polymorphisms in this locus associated with TNF-alpha production offer the opportunity of detecting new genes associated with sarcoidosis and perhaps of defining disease-associated haplotypes that bear the potential of serving as predictive markers for this disease.

Chromatin preferences of the perichromosomal layer constituent pKi-67.

The proliferation-associated nuclear protein pKi-67 relocates from the nucleolus to the chromosome surface during the G2/M transition of the cell cycle and contributes to the formation of the 'perichromosomal layer'. We investigated the in-vivo binding preferences of pKi-67 for various chromatin blocks of the mitotic chromosomes from the human and two mouse species, Mus musculus and M. caroli. All chromosomes were decorated with pKi-67 but displayed a gap of pKi-67 decoration in the centromere and NOR regions. pKi-67 distribution in a rearranged mouse chromosome showed that the formation of the centromeric gap was controlled by the specific chromatin in that region. While most chromatin served as a substrate for direct or indirect binding of pKi-67, we identified three types of chromatin that bound less or no pKi-67. These were: (1) the centromeric heterochromatin defined by the alpha satellite DNA in the human, by the mouse minor satellite in M. musculus and the 60- and 79-bp satellites in M. caroli; (2) the pericentromeric heterochromatin in M. musculus defined by the mouse major satellite, and (3) NORs in the human and in M. musculus defined by rDNA repeats. In contrast, the conspicuous blocks of pericentromeric heterochromatin in human chromosomes 1, 9 and 16 containing the 5-bp satellite showed intense pKi-67 decoration. The centromeric gap may have a biological significance for the proper attachment of the chromosomes to the mitotic spindle. In this context, our results suggest a new role for centromeric heterochromatin: the control of the centromeric gap in the perichromosomal layer.