Search

Preorganized tripodal ligands such as the N-picolyl derivatives of cis,cis-1,3,5-triamino-cis,cis-1,3,5-trimethylcyclohexane (Kemp's triamine) were prepared as analogues to N,N‘,N‘ ‘-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane (tachpyr) in hopes of enhancing the rate of formation and stability of the metal complexes. A tricyclic bisaminal was formed via the reduction of the Schiff base, while the tri(picolyl) derivative was synthesized via reductive amination of pyridine carboxaldehyde. Their cytotoxicities to the HeLa cell line were evaluated and directly compared to tachpyr and N,N‘,N‘ ‘-tris(2-pyridylmethyl)tris(2-aminoethyl)amine (trenpyr). Results indicate that N,N‘,N‘ ‘-tris(2-pyridylmethyl)-cis,cis-1,3,5-triamino-cis,cis-1,3,5-trimethylcyclohexane (Kemp's pyr) exhibits cytotoxic activity against the HeLa cancer cell line comparable to tachpyr (IC50 ≈ 8.0 μM). Both Kemp's pyr and tachpyr show higher cytotoxic activity over the aliphatic analogue of trenpyr (IC50 ≈ 14 μM), suggesting that the major contributor to the activity is the ligand's ability to form a stable and tight complex and that the equatorial/axial equilibrium impacting the complex formation for the cyclohexane-based ligands is not significant.

The iron chelator N,N‘,N‘ ‘-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane (tachpyr) was recently reported to display potent antitumor activity. The present study was focused on identifying an adequate bifunctional version of tachpyr as a lead compound for use in antibody-targeted iron depletion tumor therapy. Preparation of tachpyr derivatives having a side chain is reported, and their cytotoxic activity is evaluated in the HeLa cell line. The observed cytotoxity appears dependent on the functionalization site of the tachpyr employed for introducing the protein conjugation. Tachpyr derivatives 14 and 15 having a side chain introduced into the 5-position of the pyridyl ring display more potent cytotoxicity than tachpyr derivatives 7 and 8 having a side chain introduced onto one of the secondary amines. BOC-protected tachpyr derivative 14 exhibited the most potent cytotoxicity against this cancer cell line, which was reasonably comparable to the parent tachpyr. Tachpyr derivative 14 was further converted into bifunctional tachpyr 17 possessing a maleimide linker for conjugation with thiolated monoclonal antibodies (mAbs).

Iron is involved in essential biochemical reactions ranging from respiration to DNA synthesis. Consequently, iron deprivation has been proposed as a strategy for inhibition of tumor cell growth. We recently described a novel iron chelator, tachypyridine [N,N',N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane], and demonstrated that it not only inhibited growth of cultured tumor cells, but was actively cytotoxic. Here we explore the mechanisms underlying tachpyridine cytotoxicity. Using several criteria, including time-lapse video microscopy, DNA staining and TUNEL assays, tachpyridine was shown to specifically induce apoptotic cell death. Further, unlike numerous cytotoxic chemotherapeutic drugs which induce apoptosis by activating p53-dependent pathways, tachpyridine-mediated cell death did not require p53 activation. Although immunoblotting revealed rapid accumulation of p53 following treatment with tachpyridine, p21(WAF1) was not induced. Further, neither cytotoxicity nor apoptosis required p53. p53 null human lung cancer H1299 cells transfected with an ecdysone-inducible p53 exhibited equivalent sensitivity to tachpyridine in the presence and absence of p53, demonstrating the lack of requirement for p53 in an isogenic cell system. Further, time-lapse video microscopy and TUNEL assays demonstrated that both p53 null and p53 wild-type cells underwent apoptotic cell death in response to tachpyridine. In addition, in 55 human cancer cell lines the mean GI(50) of tachpyridine in cells with mutant p53 was virtually identical to the GI(50) in cells with wild-type p53. These results demonstrate that tachpyridine initiates an apoptotic mode of cell death that does not require functional p53. Since over 50% of human tumors contain a functionally defective p53 that reduces sensitivity to commonly used chemotherapeutic agents, such as etoposide and cisplatin, the ability of tachpyridine to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy.

We previously identified a major enhancer of the mouse ferritin H gene (FER-1) that is central to repression of the ferritin H gene by the adenovirus E1A oncogene (Tsuji, Y., Akebi, N., Lam, T. K., Nakabeppu, Y., Torti, S. V., and Torti, F. M. (1995) Mol. Cell. Biol. 15, 5152-5164). To dissect the molecular mechanism of transcriptional regulation of ferritin H, E1A mutants were tested for their ability to repress FER-1 enhancer activity using cotransfection with ferritin H-chloramphenicol acetyltransferase (CAT) reporter constructs. Here we report that p300/CBP transcriptional adaptor proteins are involved in the regulation of ferritin H transcription through the FER-1 enhancer element. Thus, E1A mutants that failed to bind p300/CBP lost the ability to repress FER-1, whereas mutants of E1A that abrogated its interaction with Rb, p107, or p130 were fully functional in transcriptional repression. Transfection with E1A did not affect endogenous p300/CBP levels, suggesting that repression of FER-1 by E1A is not due to repression of p300/CBP synthesis, but to E1A and p300/CBP interaction. In addition, we have demonstrated that transfection of a p300 expression plasmid significantly activated ferritin H-CAT containing the FER-1 enhancer, but had a marginal effect on ferritin H-CAT with FER-1 deleted. Furthermore, both wild-type p300 and a p300 mutant that failed to bind E1A but retained an adaptor function restored FER-1 enhancer activity repressed by E1A. Sodium butyrate, an inhibitor of histone deacetylase, mimicked p300/CBP function in activation of ferritin H-CAT and elevation of endogenous ferritin H mRNA, suggesting that the histone acetyltransferase activity of p300/CBP or its associated proteins may contribute to the activation of ferritin H transcription. Recruitment of these broadly active transcriptional adaptor proteins for ferritin H synthesis may represent an important mechanism by which changes in iron metabolism are coordinated with other cellular responses mediated by p300/CBP.

We examined the effects of human recombinant tumor necrosis factor-alpha (TNF) on human primary myoblasts. When added to proliferating myoblasts, TNF inhibited the expression of alpha-cardiac actin, a muscle-specific gene whose expression is observed at low levels in human myoblasts. TNF also inhibited muscle differentiation as measured by several parameters, including cell fusion and the expression of other muscle-specific genes, such as alpha-skeletal actin and myosin heavy chain. Muscle cells were sensitive to TNF in a narrow temporal window of differentiation. Northern (RNA) blot and immunofluorescence analyses revealed that human muscle gene expression became unresponsive to TNF coincident with myoblast differentiation. When TNF was added to differentiated myotubes, there was no effect on muscle gene expression. In contrast, TNF-inducible mRNAs such as interferon beta-2 still responded, suggesting that the signal mediated by TNF binding to its receptor had no effect on muscle-specific genes after differentiation.

Ferritin is a ubiquitous and highly conserved protein which plays a major role in iron homeostasis. We have identified and sequenced a full-length cDNA for murine ferritin heavy chain. The isolated cDNA is 819 nucleotides in length. It includes 546 nucleotides which encode a protein of 182 amino acids, a 5′ noncoding sequence of 120 nucleotides, and a 3′-noncoding region of 153 nucleotides. The sequence displays a high degree of homology to human ferritin H, and includes a portion of the iron-responsive element conserved in chick, frog, and human ferritin. Tumor necrosis factor (TNF), a cytokine which mediates elements of the stress response, induces expression of ferritin H mRNA. Both mouse TA1 adipocytes and human muscle cells increase expression of ferritin H mRNA 4-6-fold after 48 h exposure to TNF. This increase occurs both prior and subsequent to differentiation of adipocytes and muscle cells, and is accompanied by an increase in the synthesis of the ferritin H subunit. These findings suggest a novel role for TNF in iron metabolism.

H-kininogen is a multifunctional protein: it inhibits cysteine proteases, plays a role in contact activation of the coagulation cascade, and is the precursor of the potent proinflammatory peptide bradykinin. In the experiments described here, we identify H-kininogen as a ferritin-binding protein. Ferritin is a cellular and serum protein that is elevated in acute and chronic inflammation and many cancers. Despite numerous reports of ferritin-binding protein(s) in human serum, the nature and function of these proteins remain unclear. As a first step in characterizing the interaction between ferritin and its binding protein(s), we devised a ligand blot assay and used it to guide purification of a ferritin-binding protein from human serum. Edman degradation of the purified protein determined the sequence HNLGHGHK(H)ERDQGHG, a sequence with identity to residues 421-436 of human H-kininogen. These results were confirmed by demonstrating that commercially purified H-kininogen possessed ferritin binding activity and that ferritin binding could not be detected in plasma from kininogen-deficient individuals. Ligand blot assays mapped the ferritin binding domain to the light chain of H-kininogen chain, and revealed that both H and L recombinant ferritins possess H-kininogen binding activity. The unexpected identification of H-kininogen as a ferritin-binding protein may link ferritin in the complex chain of interactions by which H-kininogen mediates its multiple effects in contact activation and inflammation.

Ferritin is an iron-binding protein composed of two subunits, H and L. Twenty-four of these subunits assemble to form apoferritins whose subunit composition varies in a characteristic way in different tissues. Using recombinant proteins, we have assessed the role of H and L subunits in mouse ferritin function and compared these to human ferritin subunits. We report that mouse ferritin subunits exhibit considerable functional similarity to their human counterparts, including a prominent role of the H subunit in the facilitation of rapid iron uptake, and a key role of amino acid residues Glu-62 and His-65 in this process. In addition, amino acid residues important to assembly of the protein are conserved between mouse and human, permitting the formation of fully functional hybrid proteins containing both mouse and human subunits. However, murine and human ferritin H subunits also evidenced substantial functional differences; murine ferritin H showed a consistent reduction in iron uptake activity relative to human ferritin H. Creation of chimeric human/mouse ferritin H subunits by "helix swapping" mapped the domain of the protein critical to this activity difference to the DE helix. These findings suggest a novel functional role for carboxyl-terminal domains of the ferritin H subunit.

We have previously reported that the adenovirus E1A oncogene represses the transcription of the H subunit of the mouse ferritin gene. Subsequent analyses defined FER-1, a 37-nucleotide sequence located 4.1 kilobases proximal to the start site of transcription, as the target of E1A-mediated transcriptional repression and as an enhancer of the ferritin H gene. FER-1 is composed of an AP1-like sequence followed by an element with dyad symmetry. To achieve maximal enhancer activity and transcriptional repression by E1A, both elements were essential. Using gel retardation assays, we now demonstrate that the binding complex for the AP1-like sequence of FER-1 contains JunD, FosB, and ATF1. Furthermore, JunD and FosB were able to activate FER-1 enhancer activity by transient cotransfection with ferritin H-chloramphenicol acetyltransferase reporter constructs. This augmented enhancer activity was inhibited by E1A. In addition, we have defined the minimal sequence in the dyad element of FER-1 required for protein interaction. This was determined to be a C-rich sequence to which Sp1 and Sp3 bind. Experiments with recombinant proteins indicate that members of both transcription factor families simultaneously bind FER-1. Taken together, these results elucidate molecular mechanisms involved in the transcriptional regulation of a pivotal gene in iron metabolism and provide insights into the contribution of the Sp1 family to the activation of AP1-dependent enhancers.

Properties of the L- and H-type polypeptide subunits forming ferritin 24-mer molecules in mice were investigated, using the products of in vitro transcription and translation from the two cloned genes, and recombinant ferritin molecules (H24L0 or H0L24) produced by transformation in Escherichia coli. Several different conditions for analytical electrophoresis reproducibly show that the relative migration position of the two mouse ferritin subunits is reversed from that reported for ferritin H- and L-subunits in all other mammals; since mouse and human H-polypeptides almost co-migrate, this unusual relative mobility is due largely to novel properties of the murine L-subunit. This unusual electrophoretic property of the mouse L-subunit has led to conflicting reports about the subunit composition of natural mouse ferritin. Here, we show that the single major electrophoretic band given by liver ferritin purified from mice having a short-term iron overload matches that produced by the genetically defined L-polypeptide and that some bona fide H-subunits are also detected. In conclusion, it is reasonable to assume that, when mouse ferritin samples will be analyzed under the same conditions as those described here, the slower species will correspond to the L-type subunit. However, when dealing with ferritin from species other than human or mouse, it should be kept in mind that upon electrophoretic analysis of ferritin polypeptide, the designation of an electrophoretic band as being H- or L-type subunits will be very uncertain without corroboration from genetic, immunological, or amino acid sequencing data.

Ferritin is a ubiquitously distributed iron-binding protein that plays a key role in cellular iron homeostasis. It is composed of two subunits, termed H (heavy or heart) and L (light or liver). In fibroblasts and other cells, the cytokine tumor necrosis factor-alpha (TNF) specifically induces synthesis of the ferritin H subunit. Using nuclear run-off assays, we demonstrate that this TNF-dependent increase in ferritin H is mediated by a selective increase in ferritin H transcription. Transfection of murine fibroblasts with chimeric genes containing the 5'-flanking region of murine ferritin H fused to the human growth hormone reporter gene reveals that the cis-acting element that mediates this response is located approximately 4.8 kilobases distal to the start site of transcription. Deletion analyses delimit the TNF-responsive region to a 40-nucleotide sequence located between nucleotides -4776 and -4736, which we term FER-2. Electrophoretic mobility shift assays and site-specific mutations indicate that this region contains two independent elements: one contains a sequence that binds a member of the NF-kappa B family of transcription factors, and a second contains a novel sequence that partially conforms to the NF-kappa B consensus sequence and may bind a different member of the NF-kappa B/Rel transcription factor family. Thus, effects of an inflammatory cytokine on ferritin are mediated by a family of transcription factors responsive to oxidative stress.

Tumor necrosis factor (TNF) can inhibit the differentiation of preadipocytes to adipocytes and will revert differentiated adipocytes to the preadipocyte state. TNF is not toxic to either adipocytes or preadipocytes when used alone but is highly toxic to these cells when used in conjunction with cycloheximide, yielding virtually 100% killing within 4–6 h of treatment. A cell line (TA1 R-6) was isolated which is resistant to the combined toxic effects of TNF and cycloheximide. This cell line is stable and, unlike the parental cell line, does not morphologically differentiate to adipocytes or express adipocyte-specific mRNAs. It has a more transformed appearance and growth pattern and, while resistant to the toxic effects of TNF and cycloheximide in a 6-h assay, has become sensitive to cytotoxicity induced by TNF used alone in a 3-day assay. The adipocyte differentiation-inducing agents, dexamethasone and indomethacin, block the cytotoxicity induced by TNF alone in the TA1 R-6 line but do not block the rapid cytotoxicity of TNF and cycloheximide in the parental line. These results provide both genetic and pharmacologic evidence that there are at least two distinct or overlapping pathways by which TNF mediates its effects.

The authors interviewed 273 northern California testicular cancer cases aged 40 and under diagnosed between 1976 and 1981, their mothers, and matched peer controls and their mothers on prenatal hormone exposure and other variables. Included was a population-based substudy (1979-1981) of all interviewable cases reported to the San Francisco Bay Area Surveillance, Epidemiology, and End Results registry. They found odds ratios (OR) of from 8.3 (sons' report) to 4.5 (mothers' report) associated with cryptorchidism, but found no association with mothers' hormone exposure or diethylstilbestrol exposure in pregnancy. They also found a significant association with lower age at puberty (OR = 2.0); a marginally significant association with mothers' breast cancer (OR = 2.9, p = 0.054); and a significant protective effect of reported mononucleosis (OR = 0.6). These associations remained strong in the population-based substudy. When cases were divided by histology, strong and specific associations of earlier puberty (OR = 2.3) and mothers' breast cancer (OR = 4.4) with nonseminomatous cancer, and of reported mononucleosis (OR = 0.3) with seminomatous cancer, were found. These observations suggest that 1) prenatal exogenous hormone exposure does not account for a significant fraction of testicular cancer, 2) a cluster of "breast-cancer-like" risk factors are associated with nonseminomas, and 3) there is some genetic risk of nonseminomas.

Cultured TA1 adipocytes treated with tumor necrosis factor alpha (TNF) lose intracytoplasmic lipid and, over a period of days, come to resemble their predifferentiated progenitors (preadipocytes). To examine the extent to which this phenotypic reversion represents a return to a less differentiated cell, we examined three major characteristics that distinguish preadipocytes from adipocytes: (a) pattern of gene expression; (b) hormonal requirement for accelerated adipogenesis; and (c) pattern of protein synthesis. We found that within hours of TNF addition to adipocytes, mRNAs for genes whose expression is augmented during adipogenesis decreased to predifferentiated levels; in addition, like preadipocytes, TNF-treated adipocytes required exposure to hormones to accelerate adipogenesis. Further, the pattern of protein synthesis seen on polyacrylamide gels reverted to that seen before differentiation. Transforming growth factor-beta (TGF-beta) also caused a rapid decrease in expression of adipose genes when added to fully differentiated cells, an effect that was achieved by treatment with either TGF-beta 1 or TGF-beta 2. These effects were seen in the absence of a demonstrable proliferative response to either TNF or TGF-beta. Thus characteristics that define the "terminally" differentiated state in adipocytes are subject to modulation by environmental influences.