Induction Of Terminal Differentiation Research Articles

Winning Formulas to Fulfill Cancer Moonshot

The objective of this study was to develop winning formulas to fulfill cancer moonshot declared by President Biden on Sept. 12, 2022, the 60th anniversary of the moonshot speech of President Kennedy. The intention of President Biden was to use the spirit of successful moonshot project to salvage the failure of war on cancer project declared by President Nixon in 1971 [1]. Cancer therapies based on killing of cancer cells were the choice of cancer establishments in the past. The choice of killing cancer cells is understandable because the perpetual replication of cancer cells constitutes the most outstanding feature of cancer. Cancer is contributed by multiple factors. Factors other than replication of cancer cells also play essential roles on the development of cancer. Cancer arises as a consequence of wound not healing properly due to the collapse of chemo-surveillance, thus allowing Progenitor Stem Cells (PSCs) to evolve into Cancer Stem Cells (CSCs), and then to progress to faster growing Cancer Cells (CCs) through activation of oncogenes and/or inactivation suppressor genes. Cell killing creates more wounds to aggravate the already bad situation on the functionality of chemo-surveillance, which is an important mechanism to suppress the build-up of cells with abnormal Methylation Enzymes (MEs) that include PSCs, CSCs, and CCs. PSCs and their immediate derivatives CSCs are protected by drug resistant and anti-apoptosis mechanisms. Cytotoxic agents can wipe out CCs but cannot affect CSCs. The damage to chemo-surveillance contributed by cell killing agents allows CSCs to become dominant cells to cause he failure of cancer therapy, thus losing the war on cancer. Cell Differentiation Agent (CDA) formulations are the preparations consisting of Differentiation Inducers (DIs) and Differentiation Helper Inducers (DHIs), plus phenylacetylglutamine as an anti-cachexia agent. Such preparations are perfect cancer drugs to take out both CCs and CSCs by the induction of terminal differentiation through destabilization of abnormal MEs, and to restore the functionality of chemo-surveillance. Cancer therapies mediated by CDA formulations are the nature’s choice to combat cancer that can fulfill the goal of President Biden’s cancer moonshot. CDA formulations, however, cannot make the tumor to disappear, which can be easily accomplished by therapies based on cell killing. President Biden’s goal is very modest, requiring reduction of cancer mortality 50% in 25 years. That goal can be easily accomplished by winning formulas that include CDA formulations and therapies to eliminate or to kill CCs. We offer the following winning formulas: 1. Therapy with CDA-CSC, a preparation made up by an approved DI and an approved DHI, to eradicate CSCs and CCs by the induction of terminal differentiation and to restore the functionality of chemo-surveillance, followed by the surgical removal of the residual tumor. 2. Surgical removal of the primary tumor, followed by the administration of CDA-CSC to prevent recurrence and metastasis, and to restore the functionality of chemo-surveillance. 3. Radiotherapy, immunotherapy, or chemotherapy to kill CCs, followed by the administration of CDA-CSC to eradicate CSCs and to restore the functionality of chemo-surveillance.

In Pursuance of Differentiation Inducers to Combat Cancer via Targeting of Abnormal Methylation Enzymes

Cell differentiation agent-2 (CDA-2) was a promising hypomethylating agent approved by the Chinese FDA for the therapy of MDS in China. The active components of CDA-2 are differentiation inducers (DIs) and differentiation helper inducers (DHIs). DIs are chemicals capable of eliminating telomerase from abnormal MEs commonly found in human cancers. The major DI of CDA-2 is an organic acid without UV absorption. Without UV absorption as a guide, it was difficult to purify the DI of CDA-2 for identification. Thus, we pursued possible candidates to function as DIs in this study.
 Cancer MEs become abnormal due to association with telomerase. Naturally we sought telomerase inhibitors as possible candidates of DIs. Prostaglandin E2 (PGE2) attracted our attention because it was implicated to involve in wound healing, which is a major biological mission of progenitor stem cells (PSCs) and cancer stem cells (CSCs). Eradication of CSCs has been a major focus of our studies. Besides, PGE2 fits the description of the major DI of CDA-2.
 Induction of terminal differentiation (TD) of HL-60 cells by NBT assay was employed to evaluate the activity of chemicals as DIs. Cell growth was based on cell numbers. All-trans retinoic acid (ATRA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) are two well known DIs. ATRA displayed a wide active dosage range from 0.2 to 4.5 µM with a maximum of inducing 89% NBT+ cells at 3 µM. TPA displayed a narrow active dosage range from 0.2 to 0.6 nM with a maximum of inducing 84% NBT+ cells at 0.4 nM. BIBR1532 and bodine were the two telomerase inhibitors studied. Both were found active as DIs. BIBR1532 was active in the dosage range from 30 to 75 µM with a maximum of inducing 86% at 63 µM. Bodine was active in the dosage range from 60 to 98 µM with a maximum of inducing 80% at 98 µM. PGE2 was active in the dosage range from 20 to 70 µM with a maximum of inducing 80% at 56 µM.
 DIs at dosages not active as DIs could function as effective DHIs to other DIs. RI0.5 of BIBR1532, boldine and PGE2 as DHIs were 2.02 µM, 3.11 µM, and 0.92 µM, respectively.
 DIs alone, no matter how effective, could not induce NBT+ cells to reach 100%. 95% (89% plus 6% of blank) was the highest value achieved by ATRA. Incomplete induction of TD was the reason for frequent recurrence when ATRA was used alone in the therapy of acute promyelocytic leukemia (APL). A combination of ATRA and a DHI could induce NBT+ cells to reach 100% to avoid recurrence.

Role of miR-30a-5p and miR-26a-5p in induction of terminal differentiation in human K562 erythroleukemia cells

Background & Aim Background Plasmodium vivax (Pv) infections affect ∼300 million people every year. However, studies into this parasite species are hampered by the inability to set up a continuous, long-term in vitro blood stage culture for Pv. Establishing an in vitro “red blood cell matrix” that would allow continuous access to reticulocytes would facilitate the establishment of continuous, long-term in vitro Pv blood stage cultures and ultimately the study of parasite vulnerabilities that can be exploited in therapeutic approaches. Our study aims to establish culture conditions that allow for the differentiation of K562 cells into enucleated erythroid cells, serving as a constant source of reticulocytes for continuous, long-term Pv blood stage cultures. Methods, Results & Conclusion Materials and Methods Since the K562 cell line (K562) does not express the Duffy blood group antigen receptor (Fy), essential for Pv invasion, the Duffy FyB receptor was stably introduced into the K562 by lentiviral gene transfer. In addition, we combined different factors, which were known to enhance the terminal erythroid differentiation of K562 (knockdown of miR-30a-5p and -26a-5p, co-cultivation with macrophages, EPO, transferrin, insulin and mithramycin A). Effects were analysed by quantification of total cell numbers, surface markers and Giemsa staining. Hb expression was detected by Benzidin staining and PCR. Results K562 cells transduced with anti-miR-26a-5p and -30a-5p (C26) and stimulated with the induction cocktail showed a significantly increase in the amount of enucleated reticulocytes as compared to cells transduced with the empty vector. This is reflected in a stronger down-regulation of CD45 and up-regulation of CD71 as well as in the number of enucleated reticulocytes and the increase in hemoglobin production. CD235a expression was already higher in clone C26 prior to stimulation with the induction cocktail as compared to unstimulated K562 empty cells. The Duffy receptor expression remained stable in stimulated cells of the clone C26, while it was downregulated in K562 empty vector cells over the stimulation period. Conclusions The data show that knockdown of miR-26a-5p and -30a-5p are necessary to induce terminal differentiation of K562 cells. Further optimisation of culture conditions to enhance the yield of RBC blasts from K562 is necessary, as is the confirmation of functionality and susceptibility to Pv infection of these cells.

Comprehensive proteomic analysis of murine terminal erythroid differentiation

Murine-based cellular models have provided and continue to provide many useful insights into the fundamental mechanisms of erythropoiesis, as well as insights into the pathophysiology of inherited and acquired red cell disorders. Although detailed information on many aspects of these cell models is available, comprehensive proteomic data are lacking. This is a critical knowledge gap, as proteins are effectors of most biologic processes. To address this critical unmet need, proteomes of the murine cell lines Friend erythroleukemia (MEL), GATA1 erythroid (G1ER), and embryonic stem cell-derived erythroid progenitor (MEDEP) and proteomes of cultured murine marrow-derived erythroblasts at different stages of terminal erythroid differentiation were analyzed. The proteomes of MEDEP cells and primary murine erythroid cells were most similar, whereas those of MEL and G1ER cells were more distantly related. We demonstrated that the overall cellular content of histones does not decrease during terminal differentiation, despite strong chromatin condensation. Comparison of murine and human proteomes throughout terminal erythroid differentiation revealed that many noted transcriptomic changes were significantly dampened at the proteome level, especially at the end of the terminal differentiation process. Analysis of the early events associated with induction of terminal differentiation in MEDEP cells revealed divergent alterations in associated transcriptomes and proteomes. These proteomic data are powerful and valuable tools for the study of fundamental mechanisms of normal and disordered erythropoiesis and will be of broad interest to a wide range of investigators for making the appropriate choice of various cell lines to study inherited and acquired diseases of the erythrocyte.

Finding Friends in the Crowd: Three-Dimensional Cliques of Topological Genomic Domains.

The mammalian genome is intricately folded in a three-dimensional topology believed to be important for the orchestration of gene expression regulating development, differentiation and tissue homeostasis. Important features of spatial genome conformation in the nucleus are promoter-enhancer contacts regulating gene expression within topologically-associated domains (TADs), short- and long-range interactions between TADs and associations of chromatin with nucleoli and nuclear speckles. In addition, anchoring of chromosomes to the nuclear lamina via lamina-associated domains (LADs) at the nuclear periphery is a key regulator of the radial distribution of chromatin. To what extent TADs and LADs act in concert as genomic organizers to shape the three-dimensional topology of chromatin has long remained unknown. A new study addressing this key question provides evidence of (i) preferred long-range associations between TADs forming TAD “cliques” which organize large heterochromatin domains, and (ii) stabilization of TAD cliques by LADs at the nuclear periphery after induction of terminal differentiation. Here, we review these findings, address the issue of whether TAD cliques exist in single cells and discuss the extent of cell-to-cell heterogeneity in higher-order chromatin conformation. The recent observations provide a first appreciation of changes in 4-dimensional higher-order genome topologies during differentiation.

Development of Individualized Induced Pluripotent Stem Cells From Fibroblasts of Keloid Lesions in Patients

ObjectivePresently, interesting research related to induced pluripotent stem cells (iPSCs) is emerging. However, the development of new therapies and techniques for treatment of refractory diseases is still required in dermatology. We are exploring novel methods to provide stem cell therapy and elucidate research mechanisms underlying troublesome diseases by reprogramming iPSCs from the fibroblasts of keloid lesions from patients in vitro. MethodHere, we identified the expression of fibroblastic genes in the fibroblast derived from diseased individuals. Corresponding iPSCs were then produced by transfecting patient fibroblasts with non-modified RNA cocktails, expressing OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28 reprogramming factors. The pluripotency of these patient-derived iPSCs was identified by immunocytochemistry, real-time quantitative polymerase chain reaction, and teratoma formation in vivo in non-obese diabetic/severe combined immunodeficiency mice. ResultsAll iPSCs derived from patients significantly expressed the pluripotent transcription factors and could be expanded in vitro. Furthermore, induction of terminal differentiation in long-term culture and the capability of forming embryonic bodies to differentiate into all 3 germ layers in vivo were confirmed in immune-deficient mice. ConclusionFibroblasts from a keloid patient were successfully reprogrammed to iPSCs in vitro. This reprogramming may provide a basis for the production of individualized modified artificial skin to prevent rejections after xenogeneic skin transplantation and trauma through autologous skin transplantation. These cells can also offer a new platform for research on mechanisms underlying skin diseases and personal medical applications.

Thyroxine Increases Collagen Type II Expression and Accumulation in Scaffold-Free Tissue-Engineered Articular Cartilage.

Low collagen accumulation in the extracellular matrix is a pressing problem in cartilage tissue engineering, leading to a low collagen-to-glycosaminoglycan (GAG) ratio and poor mechanical properties in neocartilage. Soluble factors have been shown to increase collagen content, but may result in a more pronounced increase in GAG content. Thyroid hormones have been reported to stimulate collagen and GAG production, but reported outcomes, including which specific collagen types are affected, are variable throughout the literature. Here we investigated the ability of thyroxine (T4) to preferentially stimulate collagen production, as compared with GAG, in articular chondrocyte-derived scaffold-free engineered cartilage. Dose response curves for T4 in pellet cultures showed that 25 ng/mL T4 increased the total collagen content without increasing the GAG content, resulting in a statistically significant increase in the collagen-to-GAG ratio, a fold change of 2.3 ± 1.2, p < 0.05. In contrast, another growth factor, TGFβ1, increased the GAG content in excess of threefold more than the increase in collagen. In large scaffold-free neocartilage, T4 also increased the total collagen/DNA at 1 month and at 2 months (fold increases of 2.1 ± 0.8, p < 0.01 and 2.1 ± 0.4, p < 0.001, respectively). Increases in GAG content were not statistically significant. The effect on collagen was largely specific to collagen type II, which showed a 2.8 ± 1.6-fold increase of COL2A1 mRNA expression (p < 0.01). Western blots confirmed a statistically significant increase in type II collagen protein at 1 month (fold increase of 2.2 ± 1.8); at 2 months, the fold increase of 3.7 ± 3.3 approached significance (p = 0.059). Collagen type X protein was less than the 0.1 μg limit of detection. T4 did not affect COL10A1 and COL1A2 gene expression in a statistically significant manner. Biglycan mRNA expression increased 2.6 ± 1.6-fold, p < 0.05. Results of this study show that an optimized dosage of T4 is able to increase collagen type II content, and do so preferential to GAG. Moreover, the upregulation of COL2A1 gene expression and type II collagen protein accumulation, without a concomitant increase in collagens type I or type X, signifies a direct enhancement of chondrogenesis of hyaline articular cartilage without the induction of terminal differentiation.

Fbxw7-associated drug resistance is reversed by induction of terminal differentiation in murine intestinal organoid culture

Colorectal cancer (CRC) is one of the top three cancer-related causes of death worldwide. FBXW7 is a known tumor-suppressor gene, commonly mutated in CRC and in a variety of other epithelial tumors. Low expression of FBXW7 is also associated with poor prognosis. Loss of FBXW7 sensitizes cancer cells to certain drugs, while making them more resistant to other types of chemotherapies. However, is not fully understood how epithelial cells within normal gut and primary tumors respond to potential cancer therapeutics. We have studied genetically engineered mice in which the fbxw7 gene is conditionally knocked-out in the intestine (fbxw7∆G). To further investigate the mechanism of Fbxw7-action, we grew intestinal crypts from floxed-fbxw7 (fbxw7fl/fl) and fbxw7ΔG mice, in a Matrigel-based organoid (mini-gut) culture. The fbxw7ΔG organoids exhibited rapid budding events in the crypt region. Furthermore, to test organoids for drug response, we exposed day 3 intestinal organoids from fbxw7fl/fl and fbxw7∆G mice, to various concentrations of 5-fluorouracil (5-FU) for 72 hours. 5-FU triggers phenotypic differences in organoids including changing shape, survival, resistance, and death. 5-FU however, rescues the drug-resistance phenotype of fbxw7ΔG through the induction of terminal differentiation. Our results support the hypothesis that a differentiating therapy successfully targets FBXW7-mutated CRC cells.

Histamine H2 Receptor in Blood Cells: A Suitable Target for the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) consists in a cancer of early hematopoietic cells arising in the bone marrow, most often of those cells that would turn into white blood cells (except lymphocytes). Chemotherapy is the treatment of choice for AML but one of the major complications is that current drugs are highly toxic and poorly tolerated. In general, treatment for AML consists of induction chemotherapy and post-remission therapy. If no further post-remission is given, almost all patients will eventually relapse. Histamine, acting at histamine type-2 (H2) receptors on phagocytes and AML blast cells, helps prevent the production and release of oxygen-free radicals, thereby protecting NK and cytotoxic T cells. This protection allows immune-stimulating agents, such as interleukin-2 (IL-2), to activate cytotoxic cells more effectively, enhancing the killing of tumor cells. Based on this mechanism, post-remission therapy with histamine and IL-2 was found to significantly prevent relapse of AML. Alternatively, another potentially less toxic approach to treat AML employs drugs to induce differentiation of malignant cells. It is based on the assumption that many neoplastic cell types exhibit reversible defects in differentiation, which upon appropriate treatment results in tumor reprogramming and the induction of terminal differentiation. There are promissory results showing that an elevated and sustained signaling through H2 receptors is able to differentiate leukemia-derived cell lines, opening the door for the use of H2 agonists for specific differentiation therapies. In both situations, histamine acting through H2 receptors constitutes an eligible treatment to induce leukemic cell differentiation, improving combined therapies.

Induced Differentiation of Epithelioid Carcinoma Cell Lines: Evidence for Tumor Cell Quantal Mitosis

The effects of growth factors and calcium concentrations present in different culture media on induction of terminal differentiation were investigated for four different epidermoid carcinoma cell lines, Hela, KB, A431, and SCC-25, and their responses determined relative to those elicited by normal human keratinocytes subjected to these culture conditions. Differentiation status was determined cyto-chemically by a validated keratin protein staining method, and by autoradiographic analyses. Growth and differentiation promoting factors that influenced the direction of integrated control of growth and differentiation in normal human keratinocytes were found to be effective for some cell lines but not others. The factors examined were 1) high density arrest in serum-free and serum-containing media, 2) media shifts from high density culture in serum-containing media to low density growth factor-depleted or supplemented serum-free medium, and 3) the concentration of calcium in the media. The extent and degree of differentiation achieved varied among different cell lines depend on the presence or absence of serum, EGF and insulin protein growth factors. Certain growth media appear to sponsor keratin protein, cyto-chemically-detected differentiation, and evidence of quantal mitotic division in low density HeLa cell and SCC25 cell cultures. Epidermoid carcinoma cell lines retain limited capacity to commit to early stages of cell differentiation.

Abstract A1-21: NOTCH 1 and NOTCH 3 expressions for early stage of non-small cell lung cancer

Abstract Backgrounds: Notch gene Encode 300kDa single-pass transmembrane receptors. It can variously serve as an oncogene or a tumor suppressor and a repressor or inducer of terminal differentiation. It also regulate cell division, differentiation and survival of stem and/or progenital cells in wide range tissues. We examined Notch 1 and 3 to study the correlation of Notch expression and prognosis for non-small cell lung cancer. Methods: Paraffin-embedded tissue microarrays were constructed. We analyzed Notch 1 and 3 by immunohistochemical stain on surgical tumor specimens. Results: One hundred fifty-nine of 185 resected tissues for lung cancer (median age 62 years) were available for immunohistochemical stain. The types of tumor histology were adenocarcinoma;54, squamous cell carcinoma:79, others;26. 121 patients were taken lobectomy and 38 were done pneumonectomy. Notch 1 and 3 receptors were detected in 119 (74.8%) and 130 (81.7%) of total 159 (M:F=114:45) tumor tissues, respectively. Notch 1 receptors showed higher expressions on squamous cell carcinoma than non-squamous cell carcinoma (58.8% vs. 37.8%, p=0.03). However, it was not correlated with sex, smoking status or advanced stages. Five year-relapse free survival (RFS) and overall survival (OS) were 57.4 % and 59.3 % in 159 patients, respectively. Patients with any expression of Notch1 or 3 have correlated with neither RFS nor OS. 5 year OS for patients who showed Notch 3 expression were 62 % and the others were 47.8%, however, it was not statistically significant (p= 0.165). Both of their expressions were detected in 106 of 159 (66.7%) tumor tissues. Sixteen (10 %) had negative expressions in both of them. The patients with both positive expressions had the tendency of longer OS than all negative patients. Their 5 year OS were 60.8 % vs. 38.2% (p=0.068) and 5 year RFS were 56.5% vs. 49.7 % (p=0.35). Conclusions: Notch 1 showed higher expression on squamous cell carcinoma. Neither Notch 1 nor 3 have correlated with RFS or OS, but patients with both expressions had the tendency of longer overall survival. Citation Format: Eun Kyung Cho, Seung Yeon Ha, Jae Ik Lee, Junsick Hong, Sun Jin Sim, Jinny Park, Dong Bok Shin, Jae Hoon Lee. NOTCH 1 and NOTCH 3 expressions for early stage of non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-21.

Collapse of Ribosome Biogenesis Induces Rapid Erythroblast Differentiation

Genetic insults of ribosomal protein (RP) genes including heterozygous mutations in Diamond Blackfan Anemia (DBA) or RPS14 haploinsufficiency in acquired 5q- syndrome selectively impair erythropoiesis suggesting that the integrity of ribosome biogenesis (RB) is fully required for the production of 10e11erythrocytes per day in humans. Defective RB consecutive to RPL5, RPL11 mutation or RPS14 deletion is thought to induce cell cycle arrest and a various degree of apoptosis in erythroblasts (EB). However, recent data showed that, loss of RPL5/RPL11neither induces apoptosis nor arrests cell cycle, but hampers the progression through the G1 phase, in primary fibroblasts. Furthermore, RB coordinates cell cycle to cell growth for the maintenance of constant cell size. In this work, we investigate the role of RB in erythroid cell differentiation and cell size regulation.We first analyzed the renewal of ribosome during in vitro differentiation of human EB derived from cytapheresis CD34+ cells and mouse erythroblasts derived from mouse fetal liver by a pulsed SILAC (Stable Isotopic Labeling by Amino acids in Culture cell) riboproteomic assay. Ribosome biogenesis dramatically decreases with the disappearance of proEB and basophilic EB and the onset of poly- and ortho-chromatophilic EB. Importantly, inhibition of RNA polI by CX-5461 in proEB forced them to enter the final maturation steps with an increase of glycophorin A (GPA) expression. To study the effect of RPS14 heterozygous deletion on RB, UT7/EPO cell line was infected by a lentivirus containing an inducible GFP-shRNA RPS14. After a 48-h treatment with doxycyclin, Rps14 protein expression was reduced by half and sorted GFP-positive cells had an altered ribosome profile devoid of 40S small subunit or 80S entire particle. Consistently, RB inhibition induced a cell size reduction. Second, we compared RB level in cells responsive to SCF+EPO or EPO alone. RB was optimal when EB responded to SCF+EPO and this was correlated with cell size being higher in SCF+EPO-responsive cells compared to EPO-responsive cells. Both cytokines additively activate the cell growth regulator, p70S6K1. Third, inhibition of p70S6K1 by rapamycin, or a specific chemical S6K1 inhibitor significantly reduced RB as shown by a 50% decrease of ribosome renewal in pulsed-SILAC. Inhibition of RB by rapamycin led to a size reduction and to GPA acquisition, which are the features of erythroid cell differentiation.Our data shows that the collapse of RB due to the loss of c-Kit and reduced activation of p70S6K1 is a key step for cell growth inhibition and induction of terminal differentiation in human or mouse erythroblasts. DisclosuresNo relevant conflicts of interest to declare.

Premature Terminal Differentiation Protects from Deregulated Lymphocyte Activation by ITK-Syk

The development of hematopoietic neoplasms is often associated with mutations, altered gene expression or chromosomal translocations. Recently, the t(5, 9)(q33;q22) translocation was found in a subset of peripheral T cell lymphomas and was shown to result in an IL-2-inducible kinase-spleen tyrosine kinase (ITK-Syk) fusion transcript. In this study, we show that T cell-specific expression of the ITK-Syk oncogene in mice leads to an early onset and aggressive polyclonal T cell lymphoproliferation with concomitant B cell expansion and systemic inflammation by 7-9 wk of age. Because this phenotype is strikingly different from previous work showing that ITK-Syk expression causes clonal T cell lymphoma by 20-27 wk of age, we investigated the underlying molecular mechanism in more detail. We show that the reason for the severe phenotype is the lack of B-lymphocyte-induced maturation protein-1 (Blimp-1) induction by low ITK-Syk expression. In contrast, high ITK-Syk oncogene expression induces terminal T cell differentiation in the thymus by activating Blimp-1, thereby leading to elimination of oncogene-expressing cells early in development. Our data suggest that terminal differentiation is an important mechanism to prevent oncogene-expressing cells from malignant transformation, as high ITK-Syk oncogene activity induces cell elimination. Accordingly, for transformation, a specific amount of oncogene is required, or alternatively, the induction of terminal differentiation is defective.

Vitamin D supplementation-clarity required regarding treatment regimens and target plasma levels

The discovery of vitamin D at the beginning of the twentieth century was the result of pioneering work by numerous researchers investigating the role of vitamins and other factors in disease prevention. This culminated in Adolf Windaus being awarded the Nobel Prize for Chemistry in 1928 for his work on steroid chemistry and vitamins.1 Vitamin D deficiency is prevalent in healthy adults and is particularly high among black and Asian subjects living at higher altitudes. The National Health and Nutritional Examination Survey 2006 reports an overall prevalence rate of vitamin D deficiency of 41% in US adults.2 Older people and those who are institutionalized are more likely to have vitamin D deficiency and a UK survey found that only 16.2% of men and 13.2% of women aged ≥65 years had serum 25(OH)D levels ≥75 nmol/l.3 It is apparent but poorly understood that a strong association exists between socio-economic disadvantage and poor health in northern Europe and North America. Grimes4 suggests that poorer health and shorter life expectancy of the socio-economically disadvantaged in the UK may be partially explained by an association with the relative deficiency of vitamin D. Very few foods contain vitamin D2 or vitamin D3 and therefore, unlike other vitamins, humans rely primarily on sun exposure to obtain vitamin D. Vitamin D exerts its action through vitamin D receptors which are found in most cellular tissues. These receptors have a wide range of important biological actions including inhibition of cellular proliferation and induction of terminal differentiation, inhibition of angiogenesis, stimulation of insulin production, inhibition of renin production and stimulation of macrophage cathelicidin production.4 Vitamin D is therefore essential for normal calcium homeostasis but also has many other roles in cellular metabolism including modulation of cell growth, neuromuscular and immune function and reduction of …

Rescue of the Transcription Factors Sp1 and NFI in Human Skin Keratinocytes through a Feeder-Layer-Dependent Suppression of the Proteasome Activity

Co-culturing human skin keratinocytes along with a feeder layer has proven to considerably improve their proliferative properties by delaying massive induction of terminal differentiation. Through a yet unclear mechanism, we recently reported that irradiated 3T3 (i3T3) fibroblasts used as a feeder layer increase the nuclear content of Sp1, a positive transcription factor (TF) that plays a critical role in many cellular functions including cell proliferation, into both adult skin keratinocytes and newborn skin keratinocytes. In this study, we examined the influence of i3T3 on the expression and DNA binding of NFI, another TF important for cell proliferation and cell cycle progression, and attempted to decipher the mechanism by which the feeder layer contributes at maintaining higher levels of these TFs in skin keratinocytes. Our results indicate that co-culturing both adult skin keratinocytes and newborn skin keratinocytes along with a feeder layer dramatically increases glycosylation of NFI and may prevent it from being degraded by the proteasome.

Abstract A7: NOTCH 1 and NOTCH 3 expressions for early stage of non-small cell lung cancer

Abstract Backgrounds: Notch gene Encode 300kDa single-pass transmembrane receptors. It can variously serve as an oncogene or a tumor suppressor and a repressor or inducer of terminal differentiation. It also regulates cell division, differentiation and survival of stem and/or progenital cells in wide range tissues. We examined Notch 1 and 3 to study the correlation of Notch expression and prognosis for non-small cell lung cancer. Methods: Paraffin-embedded tissue microarrays were constructed. We analyzed Notch 1 and 3 by immunohistochemical stain on surgical tumor specimens. Results: Results: One hundred fifty-nine of 185 resected tissues for lung cancer (median age 62 years) were available for immunohistochemical stain. The types of tumor histology were adenocarcinoma; 54, squamous cell carcinoma:79, others;26. 121 patients were taken lobectomy and 38 were done pneumonectomy. Notch 1 and 3 receptors were detected in 119 (74.8%) and 130 (81.7%) of total 159 (M:F=114:45) tumor tissues, respectively. Notch 1 receptors showed higher expressions on squamous cell carcinoma than non-squamous cell carcinoma (58.8% vs. 37.8%, p=0.03). However, it was not correlated with sex, smoking status or advanced stages. Five year-relapse free survival (RFS) and overall survival (OS) were 57.4 % and 59.3 % in 159 patients, respectively. Patients with any expression of Notch1 or 3 have correlated with neither RFS nor OS. 5 year OS for patients who showed Notch 3 expression were 62 % and the others were 47.8%, however, it was not statistically significant (p= 0.165). Both of their expressions were detected in 106 of 159 (66.7%) tumor tissues. Sixteen (10 %) had negative expressions in both of them. The patients with both positive expressions had the tendency of longer OS than both negative patients. Their 5 year OS were 60.8 % vs. 38.2% (p=0.068) and 5 year RFS were 56.5% vs. 49.7 % (p=0.35). Conclusions: Notch 1 showed higher expression on squamous cell carcinoma. Neither Notch 1 nor 3 have correlated with RFS or OS, but patients with both expressions had the tendency of longer overall survival.

Highlights of This Issue

Tumor-associated myeloid cells can drive tumor progression in mice and human patients. Tumor microenvironment cues such as TNFα are capable of educating myeloid cells to transdifferentiate into a tumor-promoting population. Li and colleagues explored the mechanism by which TNFα mediates monocyte transdifferentiation into endothelial-like cells. Their work indicated that TNFα treatment of monocytes significantly increased expression of α5β1 integrin, a major fibronectin receptor. Fibronectin was a robust facilitator of endothelial differentiation of blood mononuclear cells. Blocking the interaction between α5β1 integrin and fibronectin reduced transdifferentiation both in vitro and in vivo. These findings implicate potential drug targets for anticancer treatment.Maspin, a novel serine protease inhibitor with tumor suppressive activity, has been shown to act as an endogenous inhibitor of histone deacetylase 1 (HDAC1). In the current study, Li and colleagues explored the possibility that maspin may also block or reverse DNA methylation-dependent gene silencing because histone deacetylation and DNA methylation can be mechanistically coupled. Data on the transcription regulation of glutathione s-transferase pi isoform in prostate cancer cell lines support this hypothesis and reveals a critical role of maspin in blocking pathologic DNA silencing in tumor progression.It is generally accepted that TGFβ1 is a growth inhibitor for early stages of epithelial cancer and that genetic inhibition of this pathway accelerates progression. Using primary keratinocytes expressing an inducible RAS oncogene, however, Markell and coworkers found that the response to pharmacologic inhibition of ALK5, the TGFβ type I receptor, distinguished two populations of keratinocytes linked to either induction of terminal differentiation or increased proliferation and escape from senescence. These results suggest that TGFβ1 signaling has divergent effects on subpopulations of RAS-initiated keratinocytes with potentially distinct tumorigenic potential.Targeting receptor tyrosine kinases (RTK)can yield successful cancer therapeutics. RTKs capture arrays revealed that receptors including TYRO3, AXL, MERTK, EPHB2, MET, IGF1R, KIT, FGFR3, and HER3 are activated in a panel of early passage melanoma cell lines. Transcriptional analysis suggested that some RTKs, such as FGFR3, are activated via an autocrine loop. shRNA knockdown of a subset of receptors (IGF1R, HER3, and AXL) reduced melanoma cell proliferation and migration, regardless of NRAS/BRAF mutational status. This study reveals the landscape of RTK activations in melanoma and suggests new candidates for therapeutic targeting irrespective of NRAS and BRAF driver mutations.

Valproic acid: Growth inhibition of head and neck cancer by induction of terminal differentiation and senescence

There are limited studies on the effects of drugs that modulate epigenetic regulation for head and neck squamous cell carcinoma (HNSCC). This study determined the effect of valproic acid (VPA) on HNSCC. Growth inhibition effects of VPA alone or in combination with 5-aza-2'deoxycytidine (5-aza-dC) or all-trans retinoic acid (ATRA) was evaluated with MTT and clonogenic assays on 5 HNSCC cell lines. The mechanism of growth inhibition was investigated by looking at markers of terminal differentiation and senescence. Growth inhibition profiles of HNSCC cell lines varied in response to VPA. Inhibition of clonogenic survival in response to VPA was associated with an upregulation of p21, expression of terminal differentiation markers, and cellular senescence. Notably, a combination treatment of 5-Aza-dC-VPA-ATRA enhanced growth inhibition in cells resistant to VPA. VPA is a potent inhibitor of proliferation in some HNSCC cell lines, and may be used to treat HNSCC.

Valproic acid: Growth inhibition of head and neck cancer by induction of terminal differentiation and senescence

Valproic acid: Growth inhibition of head and neck cancer by induction of terminal differentiation and senescence

Terminal differentiation of chronic myelogenous leukemia cells is induced by targeting of the MUC1-C oncoprotein

Chronic myelogenous leukemia (CML) is caused by expression of the Bcr-Abl fusion protein in hematopoietic stem cells. The MUC1-C oncoprotein is expressed in CML blasts and stabilizes Bcr-Abl. The present studies demonstrate that treatment of KU812 and K562 CML cells with GO-201, a cell-penetrating peptide inhibitor of MUC1-C oligomerization, downregulates Bcr-Abl expression and inhibits cell growth. In concert with decreases in Bcr-Abl levels, KU812 and K562 cells responded to GO-201 with induction of a differentiated myeloid phenotype as evidenced by increased expression of CD11b, CD11c and CD14. The results also show that the GO-201-treated cells undergo a late apoptotic/necrotic response, consistent with induction of terminal differentiation. Primary CML blasts expressing MUC1 similarly responded to GO-201 with induction of a more differentiated phenotype and late apoptosis/necrosis. In addition, treatment of KU812 xenografts in nude mice was associated with upregulation of CD11 and tumor regression. These findings indicate that CML blasts respond to targeting of the MUC1-C oncoprotein with induction of terminal differentiation.