Olga V. Sazonova

Family of hemorphins: co-relations between amino acid sequences and effects in cell cultures.

Hemorphins, i.e. endogenous fragments of beta-globin chain segment (32-41) LVVYPWTQRY(F) suppress the growth of transformed murine fibroblasts L929 cell culture, the effect is due to cytotoxicity and inhibition of cell proliferation. The contribution of cytotoxicity depends on the presence of Leu(32): VV-hemorphins, except VV-hemorphin-4, exhibit cytotoxicity significantly higher than respective LVV-hemorphins. Decrease of cell number induced by hemorphins depend on the extent of N- and C-terminal degradation of hemorphins: VV-hemorphins in most cases are more active than LVV-, V-hemorphins, and hemorphins. In the group of VV-hemorphins the activity of VV-hemorphin-5 (valorphin) is significantly higher than of VV-hemorphin-7, VV-hemorphin-6, and VV-hemorphin-4, meaning that the presence of C-terminal Gln is important for suppressing of cell number. The amino acid sequence VVYPWTQ corresponding to valorphin was identified as important for manifestation of the both cytotoxic and antiproliferative effects.

Antitumor effect of valorphin in vitro and in vivo: combined action with cytostatic drugs.

The action of the cytostatic drugs (epirubicin and vincristine) in combination with the endogenous antiproliferative £]-hemoglobin fragment (33-39), valorphin, was studied in tumor (L929 and A549) cell cultures, primary culture of murine bone marrow cells and in murine model of breast carcinoma in vivo. Simultaneous application of 1 µM valorphin and 1 µM epirubicin, in vitro, did not result in an additive suppressive effect on cell culture growth. Additive effects were achieved with alternating applications of the peptide and the drugs, namely, 0.5 µM (but not 1 µM) epirubicin added 24 h prior to 1 µM valorphin; 1 µM valorphin added 48 h prior to 0.1 µM epirubicin, or 0.1 µM vincristine, or 0.05 µM vincristine, which resulted in 100% cell death in the both series with vincristine and up to 78% cell biomass reduction in the experiments with epirubicin. In the in vivo model (female BLRB mice with subcutaneously inoculated syngeneic mammary carcinoma), simultaneous treatment with 25 mg/m2 epirubicin and 1 mg/kg valorphin resulted in 42% of tumor growth inhibition, as compared with the negative control group and 22% inhibition as compared with the epirubcin-treated group (at 20th day of treatment). Survival was significantly improved (69% compared to 39% in the group treated with epirubicin only) at day 26 after the treatment beginning.

Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

Retinal progenitor cells (RPCs) are neural stem cells able to differentiate into any normal adult retinal cell type, except for pigment epithelial cells. Retinoic acid (RA) is a powerful growth/differentiation factor that generally causes growth inhibition, differentiation and/or apoptosis. In this study, we demonstrate that RA not only affects mouse RPC differentiation but also improves cell survival by reducing spontaneous apoptotic rate without affecting RPC proliferation. The enhanced cell survival was accompanied by a significant upregulation of the expression of protein kinase A (PKA) and several protein kinase C (PKC) isoforms. Treatment of cells grown in RA-free media with 8-bromoadenosine3′,5′-cyclic monophosphate, a known activator of PKA, resulted in an anti-apoptotic effect similar to that caused by RA; whereas the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride led to a significant (∼32%) increase in apoptosis. In contrast, treatment of RPCs with any of two PKC selective inhibitors, 2,2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanol dimethyl ether and bisindolylmaleimide XI, led to diminished apoptosis; while a PKC activator, phorbol 12-myristate 13-acetate, increased apoptosis. These and other data suggest that the effect of RA on RPC survival is mostly due to the increased anti-apoptotic activity elicited by PKA, which might in turn be antagonized by PKC. Such a mechanism is a new example of tight regulation of important biological processes triggered by RA. Although the detailed mechanisms remain to be elucidated, we provide evidence that the pro-survival effect of RA on RPCs is not mediated by changed expression of p53 or bcl-2, and appears to be independent of β-amyloid, Fas ligand, TNF-α, ganglioside GM1 and ceramide C16-induced apoptotic pathways.

Stimulation of fibroblast proliferation by neokyotorphin requires Ca2+ influx and activation of PKA, CaMK II and MAPK/ERK

Neokyotorphin [TSKYR, hemoglobin α‐chain fragment (137–141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8‐Br‐cAMP, but not the PKC activator 4β‐phorbol 12‐myristate, 13‐acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+L‐type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester, kinase inhibitors H‐89 (PKA), KN‐62 (Ca2+/calmodulin‐dependent kinase II) and PD98059 (mitogen‐activated protein kinase). The proliferative effect of 8‐Br‐cAMP was also suppressed by KN‐62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP‐like action for neokyotorphin.

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor.

It is shown that neokyotorphin (the alpha-globin fragment 137-141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.

Proliferative Activity Of Neokyotorphinrelated Hemoglobin Fragments In Cell Cultures

alpha-Hemoglobin fragments alpha-(133-141), alpha-(134-141), alpha-(135-141), alpha-(137-141), alpha-(134-140), alpha-(133-138), alpha-(134-140) and alpha-(137-138) stimulate L929 tumor cell proliferation, alpha-(134-141) being the most active. alpha-(134-141) stimulates proliferation of M3 melanoma cells, murine embryonic fibroblasts, primary cultures of red bone marrow and spleen cells. In L929 cells the effect of alpha-(134-141) is cell density independent; in M3 cells alpha-(137-141) and alpha-(134-141) are most active at density 10,000 cells/well (96 well plate) independently on FBS content.

Fragments of functional proteins in a primary culture of human erythrocytes

According to the previously reported data, the superntant of the primary culture of human erythrocytes contains 33 hemoglobin fragments. An analysis of the supernatant of a 20% (v/v) suspension of human erythrocytes allowed us to identify additionally four peptides whose precursors are cytoplasmic β-actin (two fragments), fructose diphosphate aldolase B, and an unknown protein, and amino acids tyrosine and tryptophan. The composition and the content of the components of the supernatant did not depend on the age and blood group of donors. The dynamics of accumulation in the supernatant (20–80 min of incubation) of 14 hemoglobin fragments with the most reliably reproducible contents was obtained. The content of six peptides increased more than twofold between 20 and 40 min of incubation; the maximum increase in concentration was observed between 40 and 80 min (140%). The level of peptides that had the maximum concentration at the end of incubation was about 1000 pmol/ml of sedimented erythrocytes. The biological effects of the peptides identified in the supernatant of erythrocytes involve the stimulation of proliferation and hemopoiesis, suppression of proliferation, a bactericide effect, etc. These effects indicate the physiological importance of the peptide release by erythrocytes.

Peptidomics: A modern approach to biodiversity of peptides*

Murine myelomonocytes WEHI-3 and human erythroid leukemia K562 cells were shown to release peptides into the surrounding medium. Four N-terminal sequences of the peptides derived from unknown protein precursors were identified in the conditioned medium of WEHI-3 culture. Twelve N-terminal sequences of the peptides released by K562 cells were established. K562 cells were shown to release long hemoglobin α- and ε-chains fragments, as well as peptides derived from carbonic anhydrase XI, melanophilin, cadherin EGF LAG G-type receptor 2, aldolase A, melastatin 1, peptide homologous to ryanodine receptor fragment, and a few peptides derived from the proteins with unknown function. Comparative analysis of the peptides released by the intact erythroid leukemia K562 cells and the same cells after differentiation (induced by guanosine monophosphate, GMP, and accompanied by start of hemoglobin biosynthesis), has demonstrated that the level of six components, including all established hemoglobin fragments, carbonic anhydrase fragment, and the fragment of the unknown protein significantly increased in the course of differentiation. The spectrum of the peptides released by K562 cells strongly differed from that of the mature erythrocytes: (1) in contrast to the mature erythrocytes, K562 cells release long α-globin fragments and the fragment of ε-globin; (2) erythrocytes almost exclusively release the products of hemoglobin proteolysis; in the case of K562 cells the fragments of enzymes, receptors, and other functional proteins were found. So, the concentration and the content of the individual peptides released by the cells depend on the differentiation state. The comparative analysis of the dynamics of peptide secretion by human erythrocytes performed in the presence and the absence of the 3 % glucose has shown that the rate of the peptide release strongly depends on cell metabolism. In summary, peptidomic studies of cell cultures provide valuable information on the mechanisms of peptide pool formation in tissues and the whole organism.

β‐Actin‐derived peptides isolated from acidic extract of rat spleen suppress tumor cell growth

Twenty‐two fragments of β‐actin and β‐actin‐related protein were isolated from the acidic extracts of rat spleen tissue. β‐Actin fragments (75–90), (78–89), and (78–88), 0.01–1 µM, decreased live cell number of L929 murine tumor fibroblasts by 80–90%, with maximal cytotoxic effect of 30–40%. The fragments of (78–90) segment and the fragment of β‐actin‐related protein (69–77) were less active (inhibitory effect up to 55%, cytotoxic–up to 25%). Copyright

In vivo Proteolytic Hemoglobin Products as Tissue Growth Promoters

It is well known that growth factors are responsible for maintenance of cell proliferation in vitro and in vivo. At the same time, the ability to stimulate cell proliferation in vitro was demonstrated for endogenous fragments of functional proteins, defined earlier as components of tissue-specific peptide pools [1]. Neokyotorphin, the a-globin (137–141) fragment, has been shown earlier to stimulate proliferation of L929 transformed murine fibroblasts and brown preadipocytes independently on the presence of fetal bovine serum (FBS) [2–4]. We have studied the in vitro proliferative effects of the peptides corresponding to the a-globin (133–141) segment, and on the basis of the data obtained suggested their function in the organism.