MMC Cells Research Articles

Whole-embryonic identification of maternal microchimeric cell types in mouse using single-cell RNA sequencing

Even though the mother and the fetus of placental mammals are immunologically non-self with respect to one other, mutual exchange of small numbers of cells between them is known to occur. Maternal cells entering the fetus, called maternal microchimeric cells (MMc cells), are thought to be involved in different physiological phenomena, such as establishing immune tolerance, tissue repair, and the pathogenesis or deterioration of some inflammatory diseases and congenital malformations. While specific MMc cell types have been reported as associated with these phenomena, the contribution of MMc cells to these different outcomes remains unknown. As one possibility, we hypothesized that different embryos have differing repertoires of MMc cell types, leading to or biasing embryos toward different fates. To date, no studies have succeeded in identifying the MMc cell type repertoire of a single embryo. Accordingly, here, we isolated MMc cells from whole mouse embryos, determined their types, and analyzed their MMc cell type variability. By combining our previously established, whole-embryonic MMc isolation method with single-cell RNA sequencing, we successfully estimated the cell type repertoires of MMc cells isolated from 26 mouse embryos. The majority of MMc cells were immune-related cells, such as myeloid cells and granulocytes. We also detected stem cell-like MMc cells expressing proliferation marker genes and terminally differentiated cells. As hypothesized, we noted statistically significant inter-individual variation in the proportion of immune-related cells in the different embryos. We here successfully estimated MMc cell types in individual whole mouse embryos. The proportion of immune-related cells significantly differed among the individual embryos, suggesting that the variations are one of the potential mechanisms underlying the differing MMc-related physiological phenomena in offspring. These findings provide insight into cell-level epigenetics by maternal cells.

Whole embryonic detection of maternal microchimeric cells highlights significant differences in their numbers among individuals.

During pregnancy in placental mammals, small numbers of maternal cells (maternal microchimeric cells, or MMc cells) migrate into the fetus and persist decades, or perhaps for the rest of their lives, and higher frequencies of MMc cells are reported to correlate with variety of phenomena, such as immune tolerance, tissue repair, and autoimmune diseases. While detection of these MMc cells is considered in all pregnancies, their frequency differs largely according to tissue type and disease cases, and it remains unclear whether the number of MMc cells differs significantly among embryos in normal pregnancies. Here, for the first time, we developed a whole embryonic detection method for MMc cells using transgenic mice and counted live MMc cells in each individual embryo. Using this technique, we found that the number of MMc cells was comparable in most of the analyzed embryos; however, around 500 times higher number of MMc cells was detected in one embryo at the latest stage. This result suggests that the number of MMc cells could largely differ in rare cases with unknown underlying mechanisms. Our methodology provides a basis for testing differences in the numbers of MMc cells among individual embryos and for analyzing differences in MMc cell type repertoires in future studies. These data could provide a hint toward understanding the mechanisms underlying the variety of apparently inconsistent MMc-related phenomena.

Effect and mechanisms of LINC00152 knockdown on chemotherapy resistance in mitomycin-resistant gastric cancer NCI-N87/MMC cells

Effect and mechanisms of LINC00152 knockdown on chemotherapy resistance in mitomycin-resistant gastric cancer NCI-N87/MMC cells

Abstract 3893: Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice

Abstract Background: Melatonin-tamoxifen drug conjugate linked with a 5-carbon chain (C5) exhibited uterus protection in female mice, which did not occur when the same doses of unlinked melatonin and tamoxifen were co-administered. Anti-cancer actions of C5 and 4 others, with varying carbon lengths (C2, C4, C9, C15), were further tested in phenotypically diverse breast cancer (BC) cell lines—MCF-7(ER+/PR+), MMC(HER2+) and triple negatives(MDA-MB-231 and BT-549) in terms of cell viability and migration. In all BC lines, C4 and C5 exhibited superior potency and efficacy compared to C2, C9, C15 and unlinked melatonin and tamoxifen or 4-OH-tamoxifen controls. In this study, C4 and C5 were further tested for their anti-cancer effects in tamoxifen-resistant MCF-7 cells(TamR-MCF-7); their pharmacokinetic parameters were assessed in vitro and in vivo; and the mechanisms underlying C4- and C5-mediated anti-cancer actions were also determined by use of inhibitors against MEK1/2, MEK5 or PI3 kinase and western blot analysis. Results: C4 and C5 exhibited similar potency (TamR-MCF-7 IC50 = 4.22µM and 7.21µM; MCF-7 IC50 = 3.62nM and 440mM) and efficacy (TamR-MCF-7 = 83% and 81% inhibition; MCF-7= 71% and 90% inhibition) to inhibit TamR-MCF-7 cell viability as they did in MCF-7 cells. Regarding metabolism, C4 and C5 displayed similar CYP-mediated pharmacokinetic profiles as tamoxifen in both mouse and human liver microsomes where 40% loss of tamoxifen, C4 or C5 occurred by 10 min in mouse vs. 20 min in human. In vivo, the Tmax for C5 (oral) was 24h compared to 0.5h for C4 (oral) resulting in lower Cmax (8.9 ng/mL) and AUC0-24 (1.6 hr.ng/mL) for C5 compared to C4 (Cmax =102.6 ng/mL and AUC0-24 =143.7 hr.ng/mL) and lower oral bioavailability (0.11%) for C5 compared to C4 (4.52%). Regarding mechanisms, the effect of C4- or C5-mediated protein modulation was “context-specific” dependent upon BC phenotype (i.e., ER+, HER2+ or ER-/PR-/HER2-); the number of carbons linking melatonin to tamoxifen (i.e., 4 carbons for C4 or 5 carbons for C5); and the concentration (i.e., 1nM or 10µM) of C4 or C5 tested. For MCF-7, MMC and MDA- MB-231 cells, inhibition of MEK1/2, MEK5 and PI3K enhanced but did not block C4’s or C5’s anti-cancer actions; while for BT-549 cells, Bix02189 blocked C5’s effects. Also, in MCF-7 cells, pERK1/2, NF-κB and Runx2 were significantly modulated by C4 or C5; for MMCs, C4 and C5 modulated NF-κB and β1-integrin expression; for MDA-MB-231, MEK1/2 and MEK5 played a more central role possibly through co-modulation of NF-κB; and for BT-549, C5-mediated effects on pERK5 or PI3K-dependent regulation of pERK1/2 may underlie its anti-cancer actions. Conclusions: Melatonin-tamoxifen drug conjugates (C4 and C5) may be superior to tamoxifen therapy alone in a variety of cancers, including TamR BC. Citation Format: Mahmud Hasan, Thomas D. Wright, Saugat Adhikari, Mohamed A. Marzouk, Alaina Pericoloso, Kelsey Murgas, Miranda Burgman, Benton P. Miller, Ulrike Holzgrabe, Darius P. Zlotos, Robert E. Stratford, Paula A. Witt-Enderby. Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3893.

LncRNA-NR_033515 promotes proliferation, fibrogenesis and epithelial-to-mesenchymal transition by targeting miR-743b-5p in diabetic nephropathy

Diabetic nephropathy (DN) is a crucial microvascular complication of diabetes. Long non-coding RNAs (lncRNAs) participate in the occurrence and development of various diseases, but the function and regular mechanism of lncRNA-NR_033515 in DN is still unclear. In the present study, we demonstrated that the expression of NR_033515 was significantly increased in the serum of DN patients and was related to the different stages of DN. NR_033515 was also positively associated with diagnostic markers of DN (KIM-1 and NGAL). Overexpression of NR_033515 promoted proliferation, and inhibited apoptosis of MMC cells and increased the expression levels of proliferation-related genes. NR_033515 also accelerated the expression levels of fibrogenesis-related genes. TGF-β1 enhanced NR_033515-induced Epithelial-mesenchymal transition (EMT), while NR_033515 over-expression accelerated TGF-β1-induced EMT. Furthermore, we found that NR_033515 promoted cell proliferation and regulated P38, ASK1, Fibronectin, α-SMA, E-cadherin, and Vimentin expressions by miR-743b-5p. Therefore, our data indicated the potential role of NR_033515 in the proliferation, fibrogenesis and EMT in DN. NR_033515 could be a pivotal potential diagnostic and therapeutic target for the treatment of DN.

Abstract 3915: Melatonin-tamoxifen hybrid ligands and their effects on breast cancer

Abstract Background: A dual melatonin and tamoxifen hybrid ligand (HL) was developed and shown to display uterine protective actions while co-administration of the same doses of melatonin and tamoxifen unlinked increased uterine weight (US Patent 8785501). The anti-cancer actions of this HL (C5) and 4 others (C2, C4, C9 and C15)—which differed only in their number of carbon linkers—were further tested in 5 phenotypically diverse breast cancer (BC) cell lines—MCF-7 (ER+/PR+), tamoxifen resistant MCF-7 (TamR), MMC (HER2+) and triple negatives (MDA-MB-231 with a raf-1 mutation and BT549 with PTEN mutation) for their effects on cell proliferation, migration, protein expression and binding affinity to melatonin receptors (MT1Rs) and estrogen receptors (ERs). Analysis of HL effects on pERK1/2, pERK5, NF-kB, Runx2, and β1-integrin expression in each BC line and use of the MEK1/2 inhibitor (PD98059) or the MEK5 inhibitor (Bix02189) in the presence of the HLs helped elucidate their mechanism of action. Also, we determined the pharmacokinetic parameters of C4 and C5 HL using both in vitro and in vivo models. Results: All HLs demonstrated concentration-dependent inhibition of 2-[125I]-melatonin binding to MT1Rs, whereas C5 HL also showed equal binding affinity like melatonin. Only C4 and C5 HLs demonstrated equal affinity as tamoxifen or 4-OH-tamoxifen to ERs, and no concentration dependent inhibition of [125I]-estradiol/[3H]-estradiol binding occurred for C2, C9, and C15 HLs. All HLs were screened for their effects on BC cell proliferation and migration and it was determined that C4 and C5 demonstrated superior potency and efficacy compared to the other HLs (C2, C9, C15) and to co-exposure to melatonin and tamoxifen or 4-OH-tamoxifen (unlinked) for all BC cell lines including TamR MCF-7 cells. Acute (15 min) exposure to C4 and C5 HL increased pERK1/2 activity in MCF-7 cells and C5 HL increased NF-КB protein expression in MCF-7 and MDA-231 cells vs vehicle. Inhibition of MEK1/2 or MEK5 alone inhibited MMC, MDA-231 and BT549 cell migration. The addition of PD98059 enhanced C4-mediated inhibition of MCF-7 and BT549 cell proliferation and enhanced C5-mediated inhibition of MCF-7 cell proliferation and migration and BT549 cell migration. The addition of Bix02189 enhanced C4-mediated inhibition of MMC and BT549 cell proliferation and enhanced C5-mediated inhibition of MCF-7 and BT549 cell migration. Co-administration of Bix02189 and C4 HL blocked MDA-231 cell migration. C4 and C5 HLs displayed similar CYP-mediated loss profiles as tamoxifen in both mouse and human liver microsomes; including, the rate of metabolism being faster in mice vs human. Moreover, C4 HL showed better oral bioavailability than C5 HL in C57BL/6J female mice. Conclusions: Novel melatonin-tamoxifen HLs linked by 4 or 5 carbons show promise as anti-cancer drugs in phenotypically diverse BCs including triple negative and tamoxifen resistant and may display less uterotropic effects. Citation Format: Mahmud Hasan, Mohamed Akmal Marzouk, Saugat Adhikari, Thomas Wright, Benton Miller, Brianna Peckich, Spencer Yingling, Robert Stratford, Darius Zlotos, Jane Cavanaugh, Paula Witt-Enderby. Melatonin-tamoxifen hybrid ligands and their effects on breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3915.

The PTEN/PI3K/Akt signaling pathway mediates HMGB1-induced cell proliferation by regulating the NF-κB/cyclin D1 pathway in mouse mesangial cells.

Our previous experiment confirmed that high-mobility group box chromosomal protein 1 (HMGB1) was involved in the pathogenesis of Lupus nephritis (LN) by upregulating the proliferation of the mouse mesangial cell line (MMC) through the cyclin D1/CDK4/p16 system, but the precise mechanism is still unknown. Therefore, in the present study, we demonstrated that HMGB1 induced the proliferation of MMC cells in a time- and concentration-dependent manner, downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression, increased the level of Akt serine 473 phosphorylation, and induced p65 subunit nuclear translocation. The overexpression of PTEN prevented the upregulation of HMGB1-induced proliferation by blocking the activation of Akt. The knockdown of Akt by siRNA technology and blocking the nuclear factor-κB (NF-κB) pathway using pyrrolidine dithiocarbamate (PDTC) and SN50, inhibitors of NF-κB, both attenuated the HMGB1-induced proliferation by counteracting the activation of the cyclin D1. In addition, while sh-Akt partly blocked the nuclear translocation of the p65 subunit, PDTC did not affect the activation of the Akt induced by HMGB1 in MMC cells. These findings indicate that HMGB1 induced the proliferation of MMC cells by activating the PTEN/phosphoinositide-3-kinase (PI3K)/Akt/NF-κB signaling pathway.

IFN-γ induces lipogenesis in mouse mesangial cells via the JAK2/STAT1 pathway

The objective is to investigate the effect of high mobility group box-1 (HMGB1) on lipid deposition in γ-interferon (IFN-γ)-stimulated mouse mesangial cell line (MMC) and to determine whether the Janus kinase 2 and signal transducer and activator of transcription 1 (JAK2/STAT1) signaling pathway plays an important role in this process. We employed a control group, an IFN-γ stimulation group, and an IFN-γ + AG490 (JAK2 inhibitor) group. RNA interference aimed at sterol regulatory element-binding protein-1 (SREBP-1) or HMGB1 was used to investigate the effect of these proteins on IFN-γ-induced lipid deposition. Western blotting was used to detect phospho (p)-JAK2, JAK2, p-STAT1, STAT1, SREBP-1, fatty acid synthase (FAS), and HMGB1 protein expression. RT-PCR was used to detect SREBP-1, FAS, and HMGB1 mRNA. Oil Red O staining and the triglyceride assay were used to detect lipid deposition and triglyceride content. Results were as follows: 1) IFN-γ increased MMC cell lipid deposition, triglyceride content, and p-JAK2, p-STAT1, SREBP-1, and FAS expression; 2) SREBP-1 inhibition prevented FAS upregulation and attenuated IFN-γ-induced MMC cell lipid deposition and triglyceride content; 3) HMGB1 upregulated SREBP-1 and FAS mRNA and protein levels, which increased lipid deposition in MMC cells. Small interfering RNA-mediated inhibition of HMGB1 decreased SREBP-1 and FAS expression and lipid accumulation; 4) AG490 decreased upregulation of HMGB1 and p-JAK2/p-STAT1, as well as IFN-γ-induced lipogenesis. In conclusion, the JAK2/STAT1 pathway mediates IFN-γ-induced lipogenesis in MMC cells through regulation of HMGB1/SREBP-1/FAS.

Effect of albendazole therapy on susceptible and resistant Haemonchus contortus larvae in Mongolian gerbils (Meriones unguiculatus) and distribution of inflammatory cells in the stomach wall

Abstract The effect of albendazole therapy on the reduction of drugsusceptible and drug-resistant strains of Haemonchus contortus larvae on day 10 post infection (p.i.), distribution and the relative numbers of innate immunity cells — eosinophils/neutrophils and mast cells in the stomach wall of immunosupressed Mongolian gerbils on days 4/1, 7/4, 10/7 and 14/11 post infection/post therapy (p.i./p.t.) were investigated in the present study. The efficacy of albendazole was significantly lower on benzimidazole (BZ) resistant larvae (L3 and L4 stages) (58.92 %) than the efficacy on susceptible strain of larvae (94.15 %). H. contortus infection elicited strong inflammation in mucosal and submucosal layers of the stomach, where mucosal mast cells MMC) were in the highest numbers in the lamina propria mucosae on day 7/4 p.i./p.t. Reduction of larval numbers following treatment resulted in a gradual decrease of MMC and connective tissue mast cells (CTMC). The lower counts of CTMC in the submucosa were seen in gerbils infected with BZ-susceptible strain during the whole period post therapy. In case of infection with BZ-resistant strain, peroxidase containig cells (eosinophils) peaked on day 7/4 p.i./p.t., whereas infection with BZ-susceptible strain elicited massive accumulation of these cells on day 4/1 p.i./p.t., particularly in the submucosa. No marked differences in eosinophils localisation were observed between both groups after the therapy. Goblet cells were found only in the proximal parts of glandulae gastricae close to the mucosal surface and no differences in the distribution in the stomach wall of both groups of animals were observed. After therapy the higher larval counts in case of BZ-resistant strain were in the correlation with the lower decline of CTMC and eosinophils, but MMC numbers were not significantly different between both treated groups. Present data indicate that in early stage post infection, the distribution of individual innate immunity cells might be directly affected by the larvae, and that the genetic and consequently biological differences related to the resistance to benzimidazoles probably had the impact on the interactions of larvae with the different immune cells in their niche.

Transfected MDCK Cell Line with Enhanced Expression of CYP3A4 and P-Glycoprotein as a Model To Study Their Role in Drug Transport and Metabolism

The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and P-glycoprotein as an in vitro model for evaluating drug-herb and drug-drug of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 μM cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Similar studies were also carried out in the presence of 50 μM naringin and 3 μM morphine. Samples were analyzed by HPLC for drug and its CYP3A4 metabolite. PCR, qPCR and Western blot studies confirmed the enhanced expression of the proteins in the transfected cells. The Vivid CYP3A4 assay and ketoconazole inhibition studies further confirmed the presence of active protein. Apical to basal transport of cortisol was found to be 10- and 3-fold lower in MMC as compared to MDCK-WT and MDCK-MDR1 respectively. Higher amount of metabolite was formed in MMC than in MDCK-WT, indicating enhanced expression of CYP3A4. Highest cortisol metabolite formation was observed in MMC cell line due to the combined activities of CYP3A4 and P-gp. Transport of cortisol increased 5-fold in the presence of naringin in MMC and doubled in MDCK-MDR1. Cortisol transport in MMC was significantly lower than that in MDCK-WT in the presence of naringin. The permeability increased 3-fold in the presence of morphine, which is a weaker inhibitor of CYP3A4. Formation of 6β-hydroxy cortisol was found to decrease in the presence of morphine and naringin. This new model cell line with its enhanced CYP3A4 and P-gp levels in addition to short culture time can serve as an invaluable model to study drug-drug interactions. This cell line can also be used to study the combined contribution of efflux transporter and metabolizing enzymes toward drug-drug interactions.

Abstract B94: The roles of ERK1/2 and ERK5 in age-related breast cancer proliferation

Abstract The purpose of the current study is to examine the relative roles of the extracellular signal-regulated kinases 1, 2, and 5 (ERK1/2 and ERK5) and novel benzimidazole inhibitor compounds in cellular proliferation in breast tumors with aging. Breast cancer risk progressively increases with age, in part due to an accumulation of genetic mutations that occur during cellular division and/or exposure to environmental toxins. It has been estimated that over the next thirty years the number of Americans older than 65 years of age will double. With this increase in the aged population, the incidence of breast cancer will rise; therefore, it is important to examine the underlying mechanisms involved in breast tumor proliferation with aging. It has been well established that activation of the mitogen-activated protein kinase (MAPK) ERK1/2 leads to increased cellular proliferation and differentiation in cancer cells. Many of these studies rely on pharmacological inhibitors that were thought to be specific for the ERK1/2 pathways. Interestingly, recent studies demonstrate that these inhibitors also block the activation of a novel MAPK, ERK5. Indeed, an increase in ERK5 activity has been observed in mammary tumors in aged patients. However, the roles of ERK1/2 and ERK5 in breast cancer cell proliferation with aging have not been fully explored. In our studies, the transgenic MMTV-neu mouse model was utilized to examine ERK1/2 and ERK5 activity with age. MMTV-neu mice develop spontaneous mammary tumors which make them an appropriate model for the studies of human breast cancer. Tumors were extracted from young (3 mo), middle (9 mo), and old (15 mo) mice. Higher levels of ERK5 expression and activity were observed in middle- and old-aged animals as compared to their younger counterpart. Similarly, ERK1/2 expression and activation were increased with age. To further study the role of the ERK pathways on cell proliferation with aging, the BT-474 human breast cancer cell line and the MMTV-neu cell line (MMC) were utilized. Cell lysates were collected at passages 2, 12, 24 and 36 to mimic aging. At early passages ERK5 was constitutively active under basal conditions. At later passages, an age-dependent increase in ERK5 was observed. Breast cancer cell proliferation may be regulated by ERK5 exclusively or in conjunction with ERK1/2. We next investigated how novel benzimidazole compounds can act as specific inhibitors of ERK5 signaling in MMC and BT-474 cells. Overall, these studies will provide novel information about the cellular mechanisms involved in cancer cell proliferation and progression with aging. A better understanding of the ERK1/2 and ERK5 pathways in age-related mammary cell proliferation may lead to the development of preventative therapeutics or treatment possessing enhanced specificity for aging patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B94.

A trial comparing different surgery for pterygium

目的 采用翼状胬肉切除联合丝裂霉素及羊膜移植术和翼状胬肉切除联合丝裂霉素及自体角膜缘干细胞移植术,与以往传统的胬肉切除及暴露角膜缘巩膜术方法 进行对比研究.方法 将翼状胬肉随机分为3组,每组50例,A组:胬肉切除及暴露角膜缘巩膜术组50例(50眼);B组:翼状胬肉切除联合丝裂霉素及羊膜移植术组50例(50眼);C组:翼状胬肉切除联合丝裂霉素及自体角膜缘干细胞移植术组50例(50眼).术后随访3～22月,对3种不同术式预防复发进行研究.结果 A组复发10眼(20.00%),B组复发3眼(6.00%),C组复发2眼(4.00%).B组和C组与A组对比差异有统计学意义(P＜0.01),B组与C组对比差异无统计学意义(P＞0.05).结论 翼状胬肉切除联合丝裂霉素及羊膜移植术和翼状胬肉切除联合丝裂霉素及自体角膜缘十细胞移植术都能有效预防翼状胬肉复发。

Spin reorientation in the Er2−xFe14+2xSi3 single crystal studied by the Fe57 Mössbauer spectroscopy and magnetic measurements

Spin reorientation in the single crystal of Er2−xFe14+2xSi3 with x=0.25(5) has been studied in the temperature range of 4–300K by means of the magnetic measurements and Mössbauer spectroscopy on Fe57 by using the 14.41keV resonant transition. The bulk magnetic moment has been measured versus applied field up to ±8.9T along the c axis of the P63∕mmc cell at 4K. The hysteresis loop has been measured at 300K for the external field applied along the c axis. The bulk moment has been measured versus temperature in the moderate external field of 0.1T applied along the c axis. The ac susceptibility has been measured for several frequencies and amplitudes of the ac field applied along the c axis versus temperature either in the null external field or in the external field of 0.1T along the c axis. Mössbauer measurements were performed versus temperature on the powder sample and single crystal with the radiation beam oriented along the c axis. The spin reorientation from the [a−b] plane onto the c axis occurs for all iron sublattices except for iron dumbbells substituting erbium (2b) in the temperature range of 130–80K due to the domain flip mechanism. The reorientation is less perfect for the sublattices containing silicon, i.e., (12j) and (12k). A gradual second reorientation of the above sublattices occurs below 50K leading to the partial recovery of the high temperature spin structure. Iron dumbbells substituting erbium do not participate in this recovery. For temperatures below 20K, some dipolar contribution to the iron field on (4f) dumbbell sites is seen. It is probably induced by the reorientation of the erbium magnetic moments.

Reversal efficacy of SDZ-PSC833 and verapamil on drug-resistant human cervical carcinoma cells line

This article discusses the characteristics of the drug-resistant human cervical carcinoma cell line HeLa/MMC and investigates reversal effect of SDZ-PSC833(PSC833) and Verapamil (Ver) upon HeLa/MMC. Relative content of cell DNA detection, H3-TdR incorporation test and drug-resistance have been experimented, meanwhile a tumor transplant model in the nude mice for Hela/MMC has been established. Then the volume of cancer, tumor weight, curve line of growing up in tumor-bearing nude mouse and its morphological variation of carcinoma organism are observed to evaluate the reversal effect of PSC833 and VER. It has been found that: ➀ Compared with HeLa cells,subline HeLa/ MMC Cells’ drug-resistant capability against Mitomycin(MMC) or cisplatin(DDP) is as 5.02 folds or 2 folds as that of the former, respectively. The Hela/MMC cell line has characteristics of multi-drug resistance and stronger multiplication; ➁ PSC833 and VER can reverse resistance of HeLa/MMC to Mitomycin in vivo.PSC833 will be a better candidate for reversing multidrug resistant than verapamil in clinic application.

Intrinsic Tolerance in Autologous Collagen-Induced Arthritis Is Generated by CD152-Dependent CD4+ Suppressor Cells

Collagen-induced arthritis is a mouse model of rheumatoid arthritis (RA) and is commonly induced after immunization with type II collagen (CII) of a non-mouse origin. T cell recognition of heterologous CII epitopes has been shown to be critical in development of arthritis, as mice with cartilage-restricted transgenic expression of the heterologous T cell epitope (MMC mice) are partially tolerized to CII. However, the mechanism responsible for tolerance and arthritis resistance in these mice is unclear. The present study investigated the regulatory mechanisms in naturally occurring self-tolerance in MMC mice. We found that expression of heterologous rat CII sequence in the cartilage of mice positively selects autoreactive CD4(+) T cells with suppressive capacity. Although CD4(+)CD25(+) cells did not play a prominent role in this suppression, CD152-expressing T cells played a crucial role in this tolerance. MMC CD4(+) T cells were able to suppress proliferation of wild-type cells in vitro where this suppression required cell-to-cell contact. The suppressive capability of MMC cells was also demonstrated in vivo, as transfer of such cells into wild-type arthritis susceptible mice delayed arthritis onset. This study also determined that both tolerance and disease resistance were CD152-dependent as demonstrated by Ab treatment experiments. These findings could have relevance for RA because the transgenic mice used express the same CII epitope in cartilage as humans and because autoreactive T cells, specific for this epitope, are present in transgenic mice as well as in patients with RA.

Relaxin Stimulates Multiple Signaling Pathways: Activation of cAMP, PI3K, and PKCζ in THP‐1 Cells

Relaxin has been shown previously to stimulate cyclic AMP production and the activation of MAPK. We reported that phosphoinositide-3 kinase (PI3K) activity is required for biphasic stimulation of cAMP by relaxin and that relaxin treatment increased PI3K activity in THP-1 cells. A downstream target of PI3K is protein kinase C zeta (PKCzeta). Relaxin stimulated translocation of PKCzeta to the plasma membrane in THP-1, MCF-7, pregnant human myometrial (PHM1-31), and mouse mesangial (MMC) cells. PKCzeta translocation is PI3K dependent and independent of cAMP production. Pharmacological and antisense approaches, utilized to inhibit or knock down PKCzeta, resulted in a 40% inhibition of relaxin-stimulated cAMP production. The stimulation of PKCzeta by relaxin therefore is downstream of PI3K leading to increased cAMP production. To determine the role of PI3K/PKCzeta stimulation by relaxin on downstream-mediated events, we examined the increase in vascular endothelial growth factor (VEGF) gene expression by relaxin. Treatment of THP-1 or MMC cells with the PI3K inhibitor, LY294002, abolished the relaxin-mediated stimulation of VEGF transcript levels. In summary, relaxin has pleiotropic signaling effects in THP-1 cells activating ERK1/2, cAMP, PI3K, and PKCzeta. We have described a novel bifurcated pathway by which relaxin stimulates Gs alpha and PI3K/PKCzeta leading to increased cAMP production and increased VEGF gene expression. Some, but not all, of these pathways are detected in other cell lines which may cause the unique diversity of downstream responses from this interesting hormone.

Isolation and characterization of a mitomycin C-resistant variant of human colon carcinoma HT-29 cells.

To investigate the resistant mechanisms against MMC in human tumor cells, we isolated an MMC-resistant variant (HT-29/MMC) of HT-29 human colon carcinoma cells. HT-29/MMC cells showed 5-fold resistance to MMC as compared with the parental cell line but did not show cross-resistance to Adriamycin, vincristine, ACNU, bleomycin, or cisplatin. Treatment of the cells with dicoumarol, an inhibitor of DT-diaphorase, reduced the cytotoxicity of MMC in DT-diaphorase proficient HT-29 cells but not in HT-29/MMC cells. HT-29/MMC cells were 5 times more sensitive than HT-29 cells to menadione, which is detoxified by DT-diaphorase, DT-diaphorase was deficient in HT-29/MMC cells as determined by the enzyme activity and immunoblot analysis of the cytoplasmic proteins. Levels of cytochrome P-450 reductase and glutathione S-transferase, however, were comparable in both cell lines. The amount of [3H]-MMC found covalently bound to chromosomal DNA in HT-29/MMC cells was one-fourth that detected in HT-29 cells. Treatment with dicoumarol reduced the DNA-bound MMC in HT-29 cells but not in HT-29/MMC cells. These results indicate that the deficiency in DT-diaphorase, an activating enzyme of MMC, is one of the mechanisms of resistance in HT-29/MMC cells.

Isolation and characterization of cDNA from renal tubular epithelium encoding murine Rantes.

We have been interested in identifying proinflammatory molecules which might play a role in attracting monocytes and T cells to the kidney. Some of the new intercrines are potential candidates. In this report we have isolated cDNA encoding murine Rantes (MuRantes) from renal tubular epithelium (MCT cells). MuRantes is a 91 amino acid member of the -C-C- or intercrine beta subgroup of the Scy superfamily. The amino acid sequence for mature MuRantes was deduced from its coding cDNA and was found to be 90% homologous to its mature human counterpart (HuRantes). MCT epithelium expresses a single mRNA transcript for MuRantes of approximately 1100 bp. The MuRantes protein could be detected in cell lysates of MCT epithelium by western blotting and in the cytoplasm of MCT cells by immunofluorescence using a polyclonal antibody generated against HuRantes fusion protein. A search protocol using MuRantes-specific primers and cDNA amplification revealed that mRNAs for MuRantes are expressed additionally in syngeneic mesangial cells (MMC cells), whole kidney, liver, and spleen, as well as in nephritogenic antigen-specific CD4+ helper and CD8+ effector T cells. cDNA amplification studies also demonstrated a significant elevation in mRNA transcripts encoding MuRantes in response to the stimulation of MCT epithelium with TNF alpha and IL-1 alpha in culture, but not with TGF beta, gamma IFN, or IL-6. Our findings indicate that proximal tubular epithelium is an authentic source of MuRantes, and that transcripts encoding MuRantes are responsive to the modulating influence of paracrine factors having a known role in the development of parenchymal injury.

Trypanosoma cruzi: Antibody-dependent killing of bloodstream trypomastigotes by mouse bone marrow-derived mast cells and by mastocytoma cells

Two different populations of mast cells, that is, mastocytoma cells (P815) that were maintained either in vitro or in vivo, and mast cells obtained by differentiation of bone marrow precursor cells (MMC) in conditioned medium, were used as effector cells in antibody-dependent cytotoxic reactions (ADCC) against bloodstream trypomastigotes (BT) of Trypanosoma cruzi. The assay consisted of incubating effector cells with parasites that had been previously sensitized with immune mouse sera, immune IgG isotypes, or with medium. After the incubation period, the number of live BT was assessed. It was found that (a) cytotoxicity is antibody dependent; (b) the main isotypes involved are IgG 1, IgG 2a, and IgG 2b; (c) both types of mast cells (mastocytoma and MMC cells) are equally efficient in killing BT; (d) mastocytoma cells degranulated by pretreatment with compound 48 80 are still able to effect ADCC; (e) on optical microscope examination, large numbers of parasites were often seen attached to the cells, but only when anti- T. cruzi antibodies were present; and (f) on electron microscope examination, no integral or ruptured parasites were seen inside the cells. We conclude that both T dependent and T independent mast cells are capable of mediating ADCC by a mechanism that is probably not dependent on granule extrusion.