Review

Properties and usefulness of the original K-562 human myelogenous leukemia cell line

Hematopoietic stem cells are cells that differentiate into blood cell types. Although the placenta secretes hormones, proteins and other factors important for maternal/fetal health, cross-talk between placental and hematopoietic stem cells is poorly understood. Moreover, toxicant impacts on placental-hematopoietic stem cell communication is understudied. The goals of this study were to determine if factors secreted from placental cells alter transcriptomic responses in hematopoietic stem cells and if monoethylhexyl phthalate (MEHP), the bioactive metabolite of the pollutant diethylhexyl phthalate, modifies these effects.

We used K-562 and BeWo cells as in vitro models of hematopoietic stem cells and placental syncytiotrophoblasts, respectively. We treated K-562 cells with medium conditioned by incubation with BeWo cells, medium conditioned with BeWo cells treated with 10 μM MEHP for 24 h, or controls treated with unconditioned medium. We extracted K-562 cell RNA, performed RNA sequencing, then conducted differential gene expression and pathway analysis.

Relative to controls, K-562 cells treated with BeWo cell conditioned medium differentially expressed 173 genes (FDR<0.05 and fold-change>2.0), including 2.4-fold upregulatation of tropomyosin 4 (TPM4, a cytoskeletal regulator involved in processes such as cell morphology and migration) and 3.3-fold upregulatation of sphingosine-1-phosphate receptor 3 (S1PR3, a mediator of myeloid cell differentiation and inflammatory responses). Upregulated genes were enriched for pathways including stem cell maintenance, cell proliferation and immune processes. Downregulated genes were enriched for terms involved in protein translation and transcriptional regulation. MEHP treatment differentially expressed eight genes (FDR<0.05), including genes involved in lipid metabolism (e.g., Perilipin 2, fold-change: 1.4; Carnitine Palmitoyltransferase 1A, fold-change: 1.4).

K-562 cells, a model of hematopoietic stem cells, are responsive to media conditioned by placental cells, potentially impacting pathways like stem cell maintenance.

New water-soluble Zn(II) and Ni(II) complexes with three thiosemicarbazone ligands, modified by substitution (H, Me, Ph), H₂L1Cl, H₂L2Cl and H₂L3Cl were synthesized through the reaction of the ligands with chloride salts of metals. All complexes were characterized by physico-chemical techniques. Elemental analysis and X-ray crystallography confirmed that metal to ligand ratio 1:2 in nickel(II) complexes and 1:1 in zinc(II) complexes. In the mononuclear Ni-H₂L1Cl complex, the nickel(II) centre is surrounded by two ligands in distorted octahedral geometry. Both ligands are tridentate, nitrogen, sulfur, and oxygen donors (NSO), in thione form, with oxygen atom acting as phenolate in one of the ligands while in the other it coordinates as phenol. In the Zn-H₂L1Cl complex, the ligand acts as monodentate through sulfur atom in thione form. Geometry around the zinc(II) is tetrahedral, with remaining positions occupied by three chloride anions, resulting in Cl₃S environment around the metal. All of the complexes showed good solubility and stability in aqueous media. The cytotoxic effect of the complexes was measured using the MTT assay on MCF-7 and K562 cell lines, and the results showed that Zn-H₂L2Cl had lower IC₅₀ values compared to the other compounds. Flow cytometry analysis on the K562 cells showed the apoptosis cell death after treating with the Zn-H₂L2Cl. Cell cycle studies confirmed the cell cycle arrest in sub-G1 phase for Zn-H₂L2Cl.

Triphenylphosphonium (TPP) cations have been used for the development of tumor or myocardial diagnostic radiopharmaceuticals. In this work, the development of new [^99mTc][Tc(CO)₃(N,S,N)]⁺ complexes of the (N,S,N) tridentate chelator benzimidazol-2-yl-methylthioethylamine (L), and its triphenylphosphonium (TPP) cation derivative L-TPP is described. The TPP-moiety was conjugated at the N^τ-benzimidazol position of L. The reaction of the chelators L and L-TPP with a suitable precursor [Re(sol)₃(CO)₃]⁺ yielded single products of ReL⁺ and ReL-TPP²⁺. The complexes were characterized by spectroscopic methods and furthermore the structure of ReL was elucidated by X-ray crystallography. The respective ^99mTc-radiotracers were synthesized in high yield, their lipophilicity was measured and both exhibited high stability in cysteine, histidine solutions as well as in rat plasma. The in vitro cell studies in human erythroleukemia K-562 and glioblastoma U-87MG tumor cells showed that the tracer ^99mTcL⁺ exhibited significantly higher cellular uptake, while ^99mTcL-TPP²⁺ exhibited significantly higher mitochondrial accumulation. The tracers ^99mTcL⁺ and ^99mTcL-TPP²⁺ exhibited fast blood elimination and excretion via the hepatobiliary and the renal routes after intravenous administration in healthy mice. Tracer ^99mTcL⁺ exhibited higher myocardial uptake and renal excretion, while ^99mTcL-TPP²⁺ exhibited primarily hepatobiliary excretion. These data confirm the high mitochondrial accumulation of ^99mTcL-TPP²⁺ in vitro and show its potential as a candidate for tumor imaging.

Clinical success of chimeric antigen receptor (CAR) T cell immunotherapy requires the engineering of autologous T cells, which limits the broader implementation of CAR cell therapy. The development of allogeneic and universal cell products will significantly broaden their application and reduce costs. Allogeneic natural killer (NK) cells can be used for universal CAR immunotherapy. Here, we develop an alternative approach for the rapid expansion of primary NK and CAR-NK cells with superior expansion capability and in vivo cytotoxicity from various sources (including peripheral blood, cord blood, and tumor tissue). We apply a human B-lymphoblastoid cell-line 721.221 (hereinafter, 221)-based artificial feeder cell system with membrane-bound interleukin 21 (mIL-21) to propagate NK and CAR-NK cells. The expansion capability, purity, and cytotoxicity of NK cells expanded with 221-mIL-21 feeder cells are superior to that of conventional K562-mIL-21 feeder cells. RNA sequencing (RNA-seq) data show that 221-mIL-21 feeder cell-expanded NK cells display a less differentiated, non-exhausted, limited fratricidal, memory-like phenotype correlated with enriched metabolic pathways, which explains underlying mechanisms. Thus, “off-the-shelf” NK and CAR-NK cells with superior functionalities and expansion using a genetically modified 221-mIL-21 feeder cell expansion system will greatly support clinical use of NK immunotherapy.

The aim of this study was to determine if natural killer cell number (CD3⁻/CD16^±/CD56^±) and cytotoxic killing function predicts severity and frequency of infection in kidney transplant recipients. A cohort of 168 kidney transplant recipients with stable graft function underwent assessment of natural killer cell number and functional killing capacity immediately prior to entry into this prospective study. Participants were followed for 2 years for development of severe infection, defined as hospitalization for infection. Area under receiver operating characteristic (AUROC) curves were used to evaluate the accuracy of natural killer cell number and function for predicting severe infection. Adjusted odds ratios were determined by logistic regression. Fifty-nine kidney transplant recipients (35%) developed severe infection and 7 (4%) died. Natural killer cell function was a better predictor of severe infection than natural killer cell number: AUROC 0.84 and 0.75, respectively (P = .018). Logistic regression demonstrated that after adjustment for age, transplant function, transplant duration, mycophenolate use, and increasing natural killer function (odds ratio [OR] 0.82, 95% confidence interval [CI] 0.74-0.90; P < .0001) but not natural killer number (OR 0.96, 95% CI 0.93-1.00; P = .051) remained significantly associated with a reduced likelihood of severe infection. Natural killer cell function predicts severe infection in kidney transplant recipients.

Signaling proteins, including bone morphogenetic proteins (BMPs), specifically interact with heparan sulfate (HS). These interactions regulate protein distribution and function and are largely mediated by domains rich in basic amino acids. The N-terminal region of BMP2 and BMP4 contains one such domain with a typical Cardin-Weintraub (CW) motif, but it is unclear whether the same occurs in BMP5, BMP6, and BMP7 that constitute a separate evolutionary subgroup. Peptides spanning the N-terminal domain of BMP2/4 interacted with substrate-bound HS with nanomolar affinity, but peptides spanning BMP5/6/7 N-terminal domain did not. We re-examined the entire BMP5/6/7 sequences and identified a novel CW-like motif at their C terminus. Peptides spanning this domain displayed high-affinity HS binding, but corresponding BMP2/4 C-terminal peptides did not, likely because of acidic or noncharged residue substitutions. Peptides pre-assembled into NeutrAvidin tetramers displayed the same exact binding selectivity of respective monomers but bound HS with greater affinity. Tests of possible peptide biological activities showed that the HS-binding N-terminal BMP2/4 and C-terminal BMP5/6/7 peptides stimulated chondrogenesis in vitro, potentially by freeing endogenous BMPs. Thus, HS interactions appear largely ascribable to domains at opposite ends of BMP2/4 versus BMP5/6/7, reiterating the evolutionary distance of these BMP subgroups and possible functional diversification.