Krüppel-like factor 8 (KLF8) is expressed in gliomas of different WHO grades and is essential for tumor cell proliferation

Krüppel-like factor 8 (KLF8) has only recently been identified to be involved in tumor cell proliferation and invasion of several different tumor entities like renal cell carcinoma, hepatocellular carcinoma and breast cancer. In the present study, we show for the first time the expression of KLF8 in gliomas of different WHO grades and its functional impact on glioma cell proliferation. In order to get information about KLF8-mRNA regulation qPCR was performed and did not reveal any significant difference in samples (n = 10 each) of non-neoplastic brain (NNB), low-grade gliomas (LGG, WHO°II) and glioblastomas (GBM, WHO°IV). Immunohistochemistry of tissue samples (n = 7 LGG, 11 AA and 12 GBM) did not show any significant difference in the fraction of KLF8-immunopositive cells of all analyzed cells in LGG (87%), AA (80%) or GBM (89%). Tissue samples from cerebral breast cancer metastasis, meningiomas but also non-neoplastic brain demonstrated comparable relative cell counts as well. Moreover, there was no correlation between KLF8 expression and the expression pattern of the assumed proliferation marker Ki67, which showed high variability between different tumor grade (9% (LGG), 6% (AA) and 15% (GBM) of Ki67-immunopositive cells). Densitometric analysis of Western blotting revealed that the relative amount of KLF8-protein did also not differ between the highly aggressive and proliferative GBM (1.05) compared to LGG (0.93; p<0.05, studens t-test). As demonstrated for some other non-glial cancer entities, KLF8-knockdown by shRNA in U87-MG cells confirmed its functional relevance, leading to an almost complete loss of tumor cell proliferation. Selective blocking of KLF8 might represent a novel anti-proliferative treatment strategy for malignant gliomas. Yet, its simultaneous expression in non-proliferating tissues could hamper this approach.     

Elevated level of human RPA interacting protein α (hRIPα) in cervical tumor cells is involved in cell proliferation through regulating RPA transport

Replication protein A (RPA) is a eukaryotic single-stranded DNA binding protein that is essential for DNA replication, repair, and recombination, and human RPA interacting protein α (hRIPα) is the nuclear transporter of RPA. Here, we report the regulatory role of hRIPα protein in cell proliferation. Western blot analysis revealed that the level of hRIPα was frequently elevated in cervical tumors tissues and hRIPα knockdown by siRNA inhibited cellular proliferation through deregulation of the cell cycle. In addition, overexpression of hRIPα resulted in increased clonogenicity. These results indicate that hRIPα is involved in cell proliferation through regulation of RPA transport.     

Phospholipase C delta 4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells

Ca²⁺ is an important second messenger, and it is involved in many cellular processes such as cell death and proliferation. The rise in intracellular Ca²⁺ levels can be due to the generation of inositol 1,4,5-trisphosphate (InsP₃), which is a product of phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis by phospholipases C (PLCs), that leads to Ca²⁺ release from endoplasmic reticulum by InsP₃ receptors (InsP₃R). Ca²⁺ signaling patterns can vary in different regions of the cell and increases in nuclear Ca²⁺ levels have specific biological effects that differ from those of Ca²⁺ increase in the cytoplasm. There are PLCs in the cytoplasm and nucleus, but little is known about the functions of nuclear PLCs. This work aimed to characterize phenotypically the human PLCδ4 (hPLCδ4) in mesenchymal stem cells. This nuclear isoform of PLC is present in different cell types and has a possible role in proliferative processes. In this work, hPLCδ4 was found to be mainly nuclear in human adipose-derived mesenchymal stem cells (hASC). PLCδ4 knockdown demonstrated that it is essential for hASC proliferation, without inducing cell death. An increase of cells in G1, and a reduction of cells on interphase and G2/M in knockdown cells were seen. Furthermore, PLCδ4 knockdown increased the percentage of senescent cells, p16^INK4A+ and p21^Cip1 mRNAs expression, which could explain the impaired cell proliferation. The results show that hPLCδ4 is in involved in cellular proliferation and senescence in hASC.     

The in vitro growth of a cord blood–derived cell population enriched for CD34+ cells is influenced by its cell cycle status and treatment with hydroxyurea

Objective

Cell cycle plays a fundamental role in the physiology of hematopoietic stem and progenitor cells. In the present study we used a negative selection system to obtain an immature cell population—enriched for cord blood–derived CD34⁺ cells—and we determined its proliferation, expansion and differentiation patterns as a function of the cell cycle status. The effects of hydroxyurea (HU) were also assessed.

Influence of dichloroacetate (DCA) on lactate production and oxygen consumption in neuroblastoma cells: is DCA a suitable drug for neuroblastoma therapy?

Many cancer cells metabolize glucose preferentially via pyruvate to lactate instead to CO(2) and H(2)O (oxidative phosphorylation) even in the presence of oxygen (Warburg effect). Dichloroacetate (DCA) is a drug which is able to shift pyruvate metabolism from lactate to acetyl-CoA (tricarboxylic acid cycle) by indirect activation of pyruvate dehydrogenase (PDH). This can subsequently lead to an increased flow of oxygen in the respiratory chain, associated with enhanced generation of reactive oxygen species (ROS) which may cause apoptosis. In order to investigate if DCA may be suitable for neuroblastoma therapy, it was investigated on three human neuroblastoma cell lines whether DCA can reduce lactate production and enhance oxygen consumption. The data show, that DCA (in the low millimolar range) is able to reduce lactate production, but there was only a slight shift to increased oxygen consumption and almost no effect on cell vitality, proliferation and apoptosis of the three cell lines investigated. Therefore, DCA at low millimolar concentrations seems to be only of minor efficacy for neuroblastoma treatment.     

Establishment of a human fibroblast cell line producing tumor necrosis factor α (KMST-6/TNF) and growth inhibitory effects of its conditioned medium on malignant cells in culture

Summary To develop a new gene therapy model for cancer, a clonal cell line (KMST-6/TNF) which produces human tumor necrosis factor α (hTNF-α) has been developed by introducing hTNF-α cDNA into a human immortal fibroblast cell line (KMST-6). The conditioned medium (CM) of KMST-6/TNF cells inhibited the growth of various malignant human cell lines, but not that of normal human fibroblasts. Although the growth inhibitory effects of KMST-6/TNF CM were neutralized to a considerable degree by anti-TNF-α antibody, its inhibitory effects were more marked than the purified human natural TNF-α itself in the same units, suggesting that KMST-6/TNF CM contains some growth inhibitory substances other than TNF-α. However, interferons α, β, and γ were undetectable in the KMST-6/TNF CM.     

Electron microscopy evidence that cytoplasmic localization of the p16INK4A “nuclear” cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16^INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 ^INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16^INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16^INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16^INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16^INK4A cytoplasmic expression. We observed p16 ^INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16^INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16^INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16^INK4A inactivation similar to that observed in other tumor suppressor genes.     

Regulation of IGFBP6 gene and protein is mediated by the inverse expression and function of c-jun N-terminal kinase (JNK) and NFκB in a model of oral tumor cells

The aim of this study is to identify potential gene and protein targets when nuclear factor kappa B (NFκB) and c-jun N-terminal kinase (JNK) were inversely expressed in oral tumors. To determine which genes were regulated synergistically by the inverse expression of NFκB and JNK, a pathway specific microarray analysis was performed. While either inhibition of NFκB or activation of JNK alone was unable to affect the IGFBP6 gene expression in microarray analysis, concomitant increase in JNK activation in the presence of NFκB inhibition increased the expression of this gene significantly. Synergistic increase in IGFBP6 gene expression was also confirmed by RT-PCR and Northern blot analysis of transfected cells. Accordingly, the levels of IGFBP6 protein secretion rose synergistically when JNK was over-expressed in NFκB knock down cells. In addition, increased expression of JNK in the absence of NFκB resulted in a significant induction of cell death in oral tumors when either left untreated or treated with TNF-α and TPA. Moreover, when JNK was inhibited by dominant negative JNK (APF), a significant decrease in cell death could be observed in TNF-α and TPA treated NFκB knock down oral tumors. Therefore, increased induction of IGFBP6 gene or protein expression in oral tumors could be regarded as a potential predictive marker of tumor sensitivity and could be used for prognostic purposes, since a significant correlation could be observed between increased induction of apoptotic cell death and elevated levels of IGFBP6 in these tumors.     

19-Nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (MART-10) is a potent cell growth regulator with enhanced chemotherapeutic potency in liver cancer cells

The discovery that the active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)₂D] can modulate cellular proliferation and differentiation of cancer cells has led to its potential application as a chemotherapeutic agent to treat a variety of cancers. However, the use of 1α,25(OH)₂D is limited due to its lethal side effect of hypercalcemia upon systemic administration. To overcome this drawback, numerous analogs have been synthesized. In this report, we examined the anti-proliferative activity of a new analog, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)₂D₃ (MART-10), in HepG2 liver cancer cells, and studied the potential mechanisms mediating this action. We found that MART-10 exhibited approximately 100-fold greater activity than 1α,25(OH)₂D₃ in inhibiting HepG2 cell proliferation as determined by cell number counting method. MART-10 was also approximately 100-fold more potent than 1α,25(OH)₂D₃ in the upregulation of p21 and p27, that in turn arrested HepG2 cells at the G₀/G₁ phase to a greater extent. Given that no active caspase 3 was detected and treatment with 1α,25(OH)₂D₃ or MART-10 did not further increase the fractions of apoptotic and necrosis cells over the controls, the growth-inhibitory effect of 1α,25(OH)₂D₃ and MART-10 on HepG2 cells may not involve apoptosis. Overall, our findings suggest that MART-10 is a good candidate as a novel therapeutic regimen against liver cancer. Further pre-clinical studies using animal models and the subsequent human clinical trials are warranted.