Johannes Gerdes

The Ki-67 protein: from the known and the unknown.

The expression of the human Ki‐67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki‐67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0), makes it an excellent marker for determining the so‐called growth fraction of a given cell population. In the first part of this study, the term proliferation marker is discussed and examples of the applications of anti‐Ki‐67 protein antibodies in diagnostics of human tumors are given. The fraction of Ki‐67‐positive tumor cells (the Ki‐67 labeling index) is often correlated with the clinical course of the disease. The best‐studied examples in this context are carcinomas of the prostate and the breast. For these types of tumors, the prognostic value for survival and tumor recurrence has repeatedly been proven in uni‐ and multivariate analysis. The preparation of new monoclonal antibodies that react with the Ki‐67 equivalent protein from rodents now extends the use of the Ki‐67 protein as a proliferation marker to laboratory animals that are routinely used in basic research. The second part of this review focuses on the biology of the Ki‐67 protein. Our current knowledge of the Ki‐67 gene and protein structure, mRNA splicing, expression, and cellular localization during the cell‐division cycle is summarized and discussed. Although the Ki‐67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear. There are indications, however, that Ki‐67 protein expression is an absolute requirement for progression through the cell‐division cycle. J. Cell. Physiol. 182:311–322, 2000.

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.

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

BackgroundCpG-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.MethodsThe 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.ResultsWe 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.ConclusionsHere 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.

Immunoglobulin and T-cell receptor gene rearrangements in Hodgkin's disease and Ki-1-positive anaplastic large cell lymphoma: dissociation between phenotype and genotype.

We have determined the tumor cell immunophenotype and the rearrangement configuration of immunoglobulin and T-cell receptor genes in 39 cases of Hodgkins disease (HD), six HD-derived cell lines and 22 cases of Ki-1-positive anaplastic large cell lymphomas (Ki-1-ALC). Rearrangements were observed in 11/39 HD cases, 15/22 Ki-1-ALC, and all cell lines. Epstein-Barr virus DNA was found in five HD cases, one cell line, and one Ki-1-ALC. Both HD and Ki-1-ALC frequently displayed a dissociated genotypic and phenotypic maturation status, i.e. an immature genotype in association with late activation markers. We postulate that the tumor cells in many cases of HD and some cases of Ki-1-ALC may be derived from immature lymphoid cells by a transformation process that superimposes characteristics of mature activated lymphocytes on these cells.

Monoclonal antibodies in the diagnosis of Hodgkin's disease. The search for a rational panel.

A novel, comprehensive panel of monoclonal antibodies was tested in a large series of routinely processed lymph node biopsy specimens from patients with Hodgkins disease (69 cases), with the object of developing either definitive or adjunctive diagnostic criteria. B- and T-cell lymphomas and reactive states that could mimic Hodgkins disease were also assessed with the same monoclonal antibody panel. In addition to the popularly used anti-Leu-M1 (CD15), the panel included the recently produced Ber-H2 (CD30) antibody, which detects a formalin-resistant epitope of the Ki-1 antigen. The other monoclonal antibodies were directed against epithelial membrane antigen (Dako-EMA) and leukocyte common antigen (Dako-LC) (CD45), as well as B-cell (LN-1 and LN-2) and T-cell (MT1) associated antigens. The results showed clear phenotypic separation of nodular lymphocyte predominant subtype of Hodgkins disease from other subtypes. The lymphocytic and histocytic cells of nodular lymphocyte predominant Hodgkins disease were reactive for LN-1 (all cases) and anti-EMA (most cases) but negative for anti-Leu-M1 and Ber-H2. Within the other subtypes—i.e. nodular sclerosis and mixed cellularity—nearly all Reed-Steinberg cells and Hodgkins cells were positive for both anti-Leu-M1 and Ber-H2. Ber-H2 monoclonal antibody was observed to react more frequently with Reed-Sternberg cells and Hodgkins cells in Bouins- or formalin-fixed tissues. Pleomorphic T-cell lymphomas, which could mimic Hodgkins disease on morphology, created the same problem on phenotypic analysis. However, MT1 identified a significant proportion of T-cell lymphomas with Reed-Sternberg-like cells, having proven negative for Reed-Sternberg cells and Hodgkins cells in Hodgkins disease. Thus, a combination of anti-Leu-M1, Ber-H2, anti-EMA, LN-1, and MT1 monoclonal antibodies appears at present to be the most useful panel for the diagnosis and the differential diagnosis of Hodgkins disease.

Determination of the growth fraction in cell suspensions by flow cytometry using the monoclonal antibody Ki-67

A novel procedure for determining the growth fraction of cell suspensions by flow cytometry is described. This method identifies proliferating cells by binding the monoclonal antibody Ki-67 to a nuclear antigen present in all cells that are in the G1, S, G2, and M phase of the cell cycle, but not in the G0 phase. In a kinetic study of Na cell line U937 using concanavalin A for stimulation of peripheral blood mononuclear cells, a steady increase of Ki-67 positive cells evaluated by flow cytometry was observed. Simultaneously, the [3H]thymidine uptake of the ConA blasts was measured and compared to the expression of Ki-67. A linear correlation between the percentage of Ki-67 positive cells and the log transformed counts per minute was demonstrated, and staining with Ki-67 detected cell proliferation with the same sensitivity as 3H-TdR uptake. In addition, it was possible to stain Ki-67-labelled cells with a second cell marker if a second fluorescent dye coupled to an antibody was used. This provided the opportunity to define precisely the phenotype of proliferating cells. Conversely, the number of proliferating cells expressing certain preselected surface markers could be easily determined.

Growth fraction in human brain tumors defined by the monoclonal antibody Ki-67.

SummaryThe monoclonal antibody Ki-67, which reacts with cells in the active part of the cell cycle, was used to evaluate immunocytochemically the growth fraction in 22 primary brain neoplasms. The percentage of labelled cells reflected the histological grade of malignancy of each neoplasms. High percentage of Ki-67-positive cells were observed in one choroid plexus carcinoma (60%), one primary melanoma of meninges (40%), three medulloblastomas (40%–50%), one anaplastic astrocytoma and six glioblastomas (10%–40%). One ependymoma had 7% positive cells. Rare positive cells (1%) were present in one pilocytic astrocytoma and one ganglioglioma. Except one negative case, the meningiomas (five cases) had values of positivity ranging from 1% to 6%. Two acoustic schwannomas were negative. These results suggest that immunocytochemical staining with the Ki-67 may be a useful method for measuring the growth fraction in brain neoplasms.

The correlation of growth fractions with histologic grading and lymph node status in human mammary carcinoma.

The monoclonal antibody Ki‐67 reacts with a human nuclear antigen associated with cell proliferation that is expressed only in the G1, S, G2, and M phases of continuously cycling cells. This offers a simple opportunity to determine the growth fraction of tumors by immunostaining of fresh tissue. One hundred fifty‐four invasive carcinomas of the breast were used in this study. The average number of Ki‐67 positive cells was 15.3 ± 10.1% (range, 1%–48%), whereas in 41 benign lesions of the breast only 4.4 ± 2.6% (range, 1%–10%) of cells were positive. A correlation was found between growth fractions and histologic grading. On average, N+ tumors with less than four positive lymph nodes had a significantly higher growth fraction (20.4 ± 14.2%) than NO tumors (13.0 ± 9.2%), whereas N+ tumors with more than three lymph node metastases had only 17.3 ± 3.6% Ki‐67 positive cells. This method yielded similar results to those obtained by other researchers through the use of flow cytometry and thymidine labeling. Determination of growth fractions by Ki‐67 is suitable for routine use and may be useful in prognosis and in the selection of patients for various treatment modalities. Cancer 59:83–88, 1987.

Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in β-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.

To ‘B’ or not to ‘B’ a germinal center?

Abstract Under certain pathological conditions, germinal centers (GCs) form in many atypical sites, such as the stomach, lung and synovial joints. Thus, understanding the cellular and molecular interactions leading to a GC response is relevant not only for protective immunity but also for possible therapeutic strategies. Here, recent studies concerning the generation of GC reactions are reviewed.