Xylarinase: a novel clot busting enzyme from an endophytic fungus Xylaria curta

Xylarinase is a bi-functional fibrinolytic metalloprotease isolated from the culture filtrate of endophytic fungus Xylaria curta which is monomeric with a molecular mass of ～33.76?kDa. The enzyme displayed both plasmin and tissue plasminogen activator like activity under in vitro conditions. It hydrolyses Aα and Bβ chains of the fibrinogen. Optimal fibrinolytic activity of xylarinase is observed at 35?°C, pH 8. Ca²⁺ stimulated the fibrinolytic activity of xylarinase while Fe²⁺ and Zn²⁺ inhibited suggesting it to be a metalloprotease. The K_m and V_max values of xylarinase were 240.9?μM and 1.10?U/ml for fibrinogen and 246?μM and 1.22?U/ml for fibrin, respectively. Xylarinase was found to prolong the activated partial thromboplastin time and prothrombin time. The N-terminal sequence of xylarinase (SNGPLPGGVVWAG) did not show any homology with previously known fibrinolytic enzymes. Thus xylarinase is a novel fibrinolytic metalloprotease which could be possibly used as a new clot busting enzyme.     

Effect of human cell malignancy on activity of DNA polymerase ι

An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase ι (Pol ι) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg²⁺, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn²⁺, that activates homogeneous Pol ι preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg²⁺, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn²⁺ the activity of Pol ι was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol ι activity was observed in the presence of either Mn²⁺ or Mg²⁺. Manganese ions activated Pol ι in both cases, though to a different extent. In the presence of Mn²⁺ the Pol ι activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn²⁺-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol ι. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by cell division blocking that occurs in all normal cells except in testicles and in malignant cells.     

Absence of fibronectin and presence of plasminogen activator in both normal and malignant human mammary epithelial cells in culture

Primary monolayer cultures of normal and malignant human mammary epithelial cells were tested for fibronectin by indirect immunofluorescence using antisera specific for fibronectin. This protein was not detectable on either the normal or malignant epithelial cells. Similar results were obtained for normal and malignant mouse mammary epithelial cell cultures. Control normal and transformed fibroblasts exhibited the expected result: the normal cells were positive and the transformed cells were negative. With the use of supernatant fluids from the same cultures or an agar-overlay assay on viable cells, high levels of plasminogen-dependent fibrinolytic activity were detectable in both the normal and malignant mammary cells. Thus, two characteristics that distinguish normal from transformed fibroblasts do not serve as markers of malignancy in mammary epithelial/carcinoma systems.     

Transgenic Murine Dopaminergic Neurons Expressing Human Cu/Zn Superoxide Dismutase Exhibit Increased Density in Culture, but No Resistance to Methylphenylpyridinium-Induced Degeneration

Abstract: Primary dopaminergic neuronal cultures with increased superoxide dismutase (SOD) activity were established for studying the role of superoxide anion (O₂^?) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced degeneration of dopamine (DA) neurons. Mean SOD activity in cultures prepared from transgenic (human) Cu/Zn SOD (hSOD1) mice was 2.46–2.60 times greater than in cultures prepared from nontransgenic control mice. After 1 and 2 weeks in culture, the mean density of DA neurons [number of tyrosine hydroxylase-immunoreactive (TH-ir) cells per visual field] was significantly higher in cultures prepared from transgenic mice compared with those prepared from nontransgenic control mice (4.55–5.63 TH-ir neurons per field in hSOD1 cultures vs. 2.66–2.8 TH-ir neurons per field in control cultures). However, uptake of [³H]DA relative to uptake of [³H]GABA was only slightly greater in hSOD1 cultures than in normal cultures (14.1 nmol of DA/100 nmol of GABA vs. 12.1 nmol of DA/100 nmol of GABA). Resistance to MPP⁺ toxicity was not significantly different from that in normal cultures when based on density of surviving TH-ir cell bodies (EC₅₀ = 0.54 µM in hSOD1 and EC₅₀ = 0.37 µM in normal cultures). A more sensitive measure of DA neuron integrity and function ([³H]DA uptake) also failed to demonstrate increased resistance of hSOD1 cultures to the toxin (EC₅₀ = 73.7 nM in hSOD1 and EC₅₀ = 86.2 nM in controls). These results do not support the hypothesis that neurotoxicity of the active metabolite of MPTP, MPP⁺, is mediated by generation of O₂^? in the cytoplasm. Nevertheless, mesencephalic cultures with increased hSOD1 activity appear to survive better than normal control cultures in the oxidatively stressful environment of cell culture incubators, and such mesencephalic cells may be useful for cell grafting studies in animal models of Parkinson's disease.     

Cytotoxic and Antimicrobial Activity of Dehydrozingerone based Cyclopropyl Derivatives

A small series of 1‐acetyl‐2‐(4‐alkoxy‐3‐methoxyphenyl)cyclopropanes was prepared, starting from dehydrozingerone (4‐(4‐hydroxy‐3‐methoxyphenyl)‐3‐buten‐2‐one) and its O‐alkyl derivatives. Their microbiological activities toward some strains of bacteria and fungi were tested, as well as their in vitro cytotoxic activity against some cancer cell lines (HeLa, LS174 and A549). All synthesized compounds showed significant antimicrobial activity and expressed cytotoxic activity against tested carcinoma cell lines, but they showed no significant influence on normal cell line (MRC5). Butyl derivative is the most active on HeLa cells (IC₅₀ = 8.63 μm ), while benzyl one is active against LS174 and A549 cell lines (IC₅₀ = 10.17 and 12.15 μm , respectively).     

Characterization of a new human cell line derived from a xenografted embryonal carcinoma

Summary A human cell line has been established from a transplantable xenografted human testicular tumor, which, both in the original tumor and in the xenograft, exhibited the histological characteristics of an undifferentiated malignant teratoma (embryonal cell carcinoma). The cells in culture were undifferentiated by biochemical, morphological, and ultrastructural criteria, growing as small islands of cells that tended to form aggregates at high density. The cells showed some variation in chromosome number with 30 to 40% of the cells having a normal human karyotype. The cells expressed high levels of alkaline phosphatase, which by heat inactivation and inhibition studies was 40 to 50% placental type alkaline phosphatase. None of the cultures produced human chorionic gonadotrophin, alphafetoprotein, carcinoembryonic antigen, or fibronectin, although at high cell densities plasminogen activator could be detected at low levels. Cell surface studies showed that the cells shared antigens with the murine embryonal carcinoma cell line F9, expressedβ ₂-microglobulin at very low and variable levels, and bound the lectin peanut agglutinin. These studies suggest that this cell line has some of the characteristics described for murine embryonal carcinoma cell lines.     

Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy

Ex vivo expansion and manipulation of human mesenchymal stem cells are important approaches to immunoregulatory and regenerative cell therapies. Although these cells show great potential for use, issues relating to their overall nature emerge as problems in the field. The need for extensive cell quantity amplification in vitro to obtain sufficient cell numbers for use, poses a risk of accumulating genetic and epigenetic abnormalities that could lead to sporadic malignant cell transformation. In this study, we have examined human mesenchymal stem cells derived from bone marrow, over extended culture time, using cytogenetic analyses, mixed lymphocyte reactions, proteomics and gene expression assays to determine whether the cultures would retain their potential for use in subsequent passages. Results indicate that in vitro cultures of these cells demonstrated chromosome variability after passage 4, but their immunomodulatory functions and differentiation capacity were maintained. At the molecular level, changes were observed from passage 5 on, indicating initiation of differentiation. Together, these results lead to the hypothesis that human mesenchymal stem cells cultures can be used successfully in cell therapy up to passage 4. However, use of cells from higher passages would have to be analysed case by case.     

Studies on experimentally produced overgrowth in chick embryo brain,using tissue culture and transplantation techniques

Summary Experimental overgrowth was produced in chick embryo brains according to the method described earlier (1955, 1959). Pieces of overgrown mesencephalic tissue were cultured in vitro or studied with transplantations to the braincavity of host embryos.When cultured in vitro, cell strands developed in some cultures, showing differences in growth rate and capacity of fibrinolytic activity as compared with normal neural epithelium.After transplantation to the brain-cavity, one case out of six showed a well delimited tumorous formation of a very benign appearance. In the other five cases, the graft tissue had completely merged with the host tissue, showing invasion into the latter. Numerous mitoses were seen and lots of rosette formations. It is concluded that the overgrowth cells may show a strong tendency to invade brain tissue.The cost of this investigation was defrayed by a grant from the Swedish Medical Research Council.     

Effects of successivein vitro andIn vivo passages on the virulence of the entomopathogenic fungus,Nomuraea rileyi

There was no significant decrease or increase in the activity of conidia ofNomuraea rileyi after 12_{in vivo} serial passages in larvae ofTrichoplusia ni (Hübner) or after 12in vitro serial passages on a semi-synthetic medium. The LC-50 at the start of the serial passage was 16.8±4.0 conidia/mm²; after 12 serial passages the LC-50 for thein vitro- andin vivo-produced conidia was 16.2±2.5 conidia/mm² and 12.0±1.9 conidia/mm², respectively.

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Résumé Il n'y a pas d'augmentation ou de réduction significative dans l'activité des conidies deNomuraea rileyi après 12 passages en sériein vivo chez les larves deTrichoplusia ni (Hübner) ou après 12 passages en sériein vitro sur un milieu semi-synthétique. La concentration léthale 50 au début de ces passages était de 16,8±4,0 conidies/mm², après 12 passages la CL 50 des conidies produitesin vitro etin vivo fut respectivement de 16,2±2,5 conidies/mm² et de 12,0±1,9 conidies/mm².

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Mention of a proprietary product in this paper does not constitute a recommendation for use by the USDA.     

Fucosyltransferase III and sialyl-Lex expression correlate in cultured colon carcinoma cells but not in colon carcinoma tissue

The potential contribution of fucosyltransferases to the overexpression of sialyl-Le^x antigen was investigated in the colon carcinoma cell line HT-29 and in human colon carcinoma tissue. In HT-29 cells as well as in normal or malignant colonic tissues Fuc-TIII, Fuc-TIV, Fuc-TVI but not Fuc-TV nor Fuc-TVII were detectable after RT-PCR. Sodium butyrate treatment of HT-29 cells increased (to about 200%) and DMSO treatment decreased (to about 20%) the expression of sialyl-Le^x. This modulation of sialyl-Le^x was concomitant with the analogous increase/decrease of mRNA of Fuc-TIII but not Fuc-TIV. Fuc-TVI was not detectable by Northern blotting in HT-29 cells. In six human colon carcinomas which exhibited strong overexpression of sialyl-Le^x, the expression of Fuc-TIII-mRNA was the same or lower than in the corresponding normal colonic tissue. Thus Fuc-TIII expression may be affecting the expression of the sialyl-Le^x moiety in HT-29 cells but not in human colon carcinoma tissue.     

Induction of carcinoembryonic antigen expression in a three-dimensional culture system

Summary MIP-101 is a poorly differentiated human colon carcinoma cell line established from ascites that produces minimal amounts of carcinoembryonic antigen (CEA), a 180 kDa glycoprotein tumor marker, and nonspecific cross-reacting antigen (NCA), a related protein that has 50 and 90 kDa isoforms, in monolayer culture. However, MIP-101 produces CEA when implanted into the peritoneum of nude mice but not when implanted into subcutaneous tissue. We tested whether three-dimensional (3D) growth was a sufficient stimulus to produce CEA and NCA 50/90 in MIP-101 cells, because cells grow in 3D in vivo rather than in two-dimensions (2D) as occurs in monolayer cultures. To do this, MIP-101 cells were cultured on microcarrier beads in 3D cultures, either in static cultures as nonadherent aggregates or under dynamic conditions in a NASA-designed low shear stress bioreactor. MIP-101 cells proliferated well under all three conditions and increased CEA and NCA production three- to four-fold when grown in 3D cultures compared to MIP-101 cells growing logarithmically in monolayers. These results suggest that 3D growth in vitro simulates tumor function in vivo and that 3D growth by itself may enhance production of molecules that are associated with the metastatic process.     

Cleansing the long-term micropropagated triploid watermelon cultures from covert bacteria and field testing the plants for clonal fidelity and fertility during the 7–10 year period in vitro

The study was aimed primarily at cleansing the in vitro-decline displaying long-term micropropagated triploid watermelon ‘Arka Manik’ cultures from covert bacteria and their further field testing. Disinfectant treated shoots showed endophytic survival but to a lesser extent in shoot-tips. Culturing the NaOCl (5 min) treated shoot-tips on filter paper bridges in liquid watermelon medium containing single antibiotic (gentamycin (Gm), amoxycillin (Ax) or cefazolin (Cz) at 50, 250, 500 or 1000 mg l⁻¹) for 1 month followed by repeated indexing of medium and tissue for two-four subculture passages facilitated the cleansing of cultures with 12.5% recovery as monitored for 2 years. Partial bleaching damage by NaOCl, phytotoxicity to higher levels of antibiotics, poor growth response in the initial sucrose-free medium and rampant hyperhydricity came in the way of a higher recovery. The effectiveness of the above approach was ascertained after back inoculating clean cultures with a mixture of Gram-positive and Gram-negative bacteria yielding 30, 45 and 35% clean cultures in Gm, Ax and Cz (250 mg l⁻¹ for 1 month) treatments, respectively in modified medium compared with 10% recovery after mere HgCl₂ surface sterilization. The results indicated that antibiotic treatment was essential but not its choice, and extended culture-indexing subsequent to disinfection or antibiotic treatment was crucial in identifying clean stocks. Cleansed cultures, showed restoration of growth but a drop in rooting. Most of the in vitro cultures appeared normal and true-to-type during the 7–10 year period in vitro but a small proportion of bacteria-harboring stocks displayed ‘epigenetic variations’. Acclimatized plants and those in the field also appeared true-to-type but for a minor proportion derived from bacteria-harboring stocks. Field-plants which originated from bacteria-freed stocks after 9 years of continuous culturing were normal and fertile validating the possibility of keeping treasured cultures in vitro for long periods if covert contaminants are checked.     

Antiproliferative activity of morpholine-based compounds on MCF-7 breast cancer,colon carcinoma C26, and normal fibroblast NIH-3T3 cell lines and study of their binding affinity to calf thymus-DNA and bovine serum albumin

Abstract

This report describes the results of a study on the antiproliferative activity of the morpholine-based ligand 1,3-bis(1-morpholinothiocarbonyl)benzene (HL) and its nickel(II) complex (NiL) against human breast cancer cells (MCF-7), colon carcinoma cells (C26), and normal fibroblast NIH-3T3 cells. NiL showed better cytotoxicity on both cancerous cells relative to normal cells in vitro with the highest selective index of 2.22 in MCF-7 cells. The interaction of both compounds with calf thymus DNA (CT DNA) and bovine serum albumin (BSA) was studied using various spectroscopic techniques and analytical methods such as UV???vis titrations, thermal denaturation, circular dichroism, competitive fluorescent intercalator displacement assays, as well as molecular modeling. The fluorescence intensity of the probe molecule increases clearly when HL and NiL are added to the methylene blue (MB)–DNA system. Furthermore, the binding of HL and NiL quenches the BSA fluorescence, revealing a 1:1 interaction with a binding constant of about 10⁵?M^?1.

Communicated by Ramaswamy H. Sarma     

Single‐cell analysis of p16INK4a and p21WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Herein we used single‐cell observation methods to gain insight into the roles of p16^INK4A and p21^WAF1 (hereafter p16 and p21) in replicative senescence and ionizing radiation‐induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li‐Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence‐associated β‐galactosidase. In addition, proliferating early‐passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53‐proficient (normal and AT) fibroblasts. In p53‐deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation‐triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild‐type p53 activity. The possibility that one of the tumor‐suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results. J. Cell. Physiol. 223: 57–67, 2010. © 2009 Wiley‐Liss, Inc.     

In vitro augmentation of cytotoxicity by N-CWS in peritoneal polymorphonuclear leukocytes (PMNs) induced by PSK,OK-432 or N-CWS

Peritoneal polymorponuclear leukocytes (PMNs) were collected from the peritoneal cavity of C3H/He mice 6 hrs after intraperitoneal (i.p.) injection of 2.5 mg/head of PSK, 1 KE (100 µg)/head of OK-432 or 200 µg/head ofNocardia rubra cell wall skeleton (N-CWS). Withoutin vitro stimulation, these PMNs did not show cytotoxicity to syngeneic MM46 mammary carcinoma cells in⁵¹Cr release assay. Cytotoxicity of these PMNs was augmented by the addition of 25 µg/ml of N-CWS but not of PSK or OK-432 to cultures for the assay at the beginning of the culture. H₂O₂ production of PSK-induced PMNs was increased by thein vitro addition of 25 µg/ml of N-CWS but not of PSK. These results suggest that PSK as well as OK-432 and N-CWS can induce PMNs capable of responding further to N-CWS as the second stimulant.     

Antiproliferative Effect of the Tripeptide PyroGlu-Phe-GlyNH2on Murine Melanocytes: Transitory Delay of Cell Growthin Vitroand the Cell Cycle Specificity

Cell growth and differentiation in melanocyte cell populations are regulated by a wide range of bioactive substances. Recently, the tripeptide pyroGlu-Phe-GlyNH₂which inhibits melanocyte growthin vitrowas identified in both murine nontransformed melanocytes and malignant melanoma cells. The present study was undertaken to investigate the cell cycle specificity as well as the growth inhibitory profile of the tripeptide after a single or repeated administration to melanocyte cultures. Dose-related effects of the peptide were studied using three different bioassay systems: estimation of cell number, DNA synthesis, and cell flux into mitosis. Growth of melanocyte cultures as well as melanocyte mitotic activity were found to be reduced significantly by the tripeptide at two separate dose levels (10⁻¹¹and 10⁻¹⁴–10⁻¹⁵M). Growth inhibition of melanocyte population did not last long: less than 36 h after the first and less than 24 h after the second peptide addition to the cultures. The level of DNA synthesis in melanocytes remained unchanged after a single peptide administration. The findings indicate that the tripeptide pyroGlu-Phe-GlyNH₂causes transitory delay of cell growth in cultured melanocyte population resulting from a reversible inhibition of melanocyte transition from the G₂-phase of the cell cycle into mitosis.     

Fluorimetric Analysis of Phospholipase Activity in Tetrahymena pyriformis GL.

The unicellular Tetrahymena enzymatically split the synthetic phosphodiester, 4-methylum-belliferyl phosphocoline substrate. The enzyme activity was completely blocked in vitro and drastically inhibited in vivo by G-protein activating fluorides (NaF; AlF₄ ^– and BeF₃ ^–). The phospholipase A₂ inhibitor, quinacrine, and the protein phosphatase inhibitor, neomycin, inhibited the enzyme activity in vitro and activated it in vivo. Another phospholipase A₂ inhibitor 4-bromo phenacyl bromide was ineffective in vivo and in vitro alike, as well as the cyclooxygenase inhibitor indomethacin. Results of these experiments indicate that some treatments could be specific for a well defined activity (e.g., phospholipase A₂, G-protein) but subject to influence by other enzymes (e.g., phospholipase C, sphingomyelinase). The experiments call attention to the differences in the results of the in vivo and in vitro studies.     

Cellular growth modulation. I. Effects of extracellular matrix and tumor cell conditioned medium

Earlier studies reported the enzymatic modulation of the cell surface in malignant transformation of human normal mammary epithelial cells and in conversion of mammary carcinoma. Carcinoembryonic antigen (CEA) is a neoplasm-associated antigen, its production and release is used to monitor changes in cell phenotype. The present study shows that CEA production and release by human colon carcinoma (CCC), and by colon cells from patients with familial polyposia coli (FPC) and ulcerative colitis (UCC) is inhibited when the cells are cultured in contact with confluent normal colon epithelial (HNCEC) cell monolayer. Footprints left behind and/or conditioned media from HNCEC cells inhibited, whereas footprints left behind and/or conditioned media from CCC, FPC or Ucc enhanced CEA release. During sequential passages of HNCEC cells grown on footprints and/or in spent media from CCC cultures, HNCEC cells acquire the ability to produce and release CEA, and to develop tumors in athymic Nu/Nu mice. On the other hand, during sequential passages, CCC, FPC or UCC grown in spent media, or on footprints left behind HNCEC cells, showed significant decrease in CEA production and release, and in oncologic ability in athymic mice. It is concluded that both the extracellular matrix, and a growth-regulating factor(s) in the spent medium modulate cellular transformation. Quantitative data on CEA-release indicate that FPC and UCC represent an intermediary stage between normal colon epithelial cells and colon carcinoma cells, i.e. a preneoplastic stage.     

Role of Glycine in Blood Coagulation Processes

We studied hemocoagulant properties of the amino acid glycinein vitroand after intravenous administration to animals (rats). Addition of 10^–3and 10^–4M glycine to the plasma increases the aggregability of thrombocytesin vitro, while all other test concentrations had virtually no effect on hemostatic parameters of the plasma. Intravenous administration of glycine increased the functional activity of the enzymatic fibrinolytic unit of the anticoagulation system. Dose dependence of this effect has been established. The causes of these changes and possible application of glycine as an agent activating fibrinolysis are discussed.     

Cytostatic and Cytotoxic Effects of Pannon (P-30 Protein), A Novel Anticancer Agent

P-30 Protein is a novel protein, of molecular weight approximately 15 kD, obtained from the extract of a vertebrate tissue showing in vivo antitumour activity. Cytostatic and cytotoxic effects of this product in its purified form (P-30 Protein) or in partially purified extracts (Pannon) were studied in vitro on human leukaemic HL-60, human submaxillary carcinoma A-253, human colon adenocarcinoma Colo 320 CM and murine erythroleukaemia (Friend leukaemia) cell lines. of these cells, HL-60, A-253 and Colo 320 CM were sensitive and Friend leukaemia resistant to this agent. the effects were time- and concentration-dependent. During the initial 24–48 h of treatment, a slowdown in cell proliferation was apparent but cell death was not extensive. After 24–48 h, there was a reduction in the proportion of cells in S phase of the cell cycle and the cells became preferentially arrested in G₁ phase. the G₁ cells showed high heterogeneity with respect to RNA content and some cells were characterized by very low RNA content. Progressive cell death occurred in cultures maintained with Pannon for up to 7 d in proportion to its concentration. Reductions of 50 and 90% in clonogenicity of A-253 cells were observed during their growth in the presence of 0.13 and 1.5 μg/ml of this protein, respectively. Exponentially growing cells were more sensitive to Pannon compared with cells from confluent cultures. Colonies of A-253 cells growing in the presence of Pannon were much smaller in size compared with control colonies, indicating that the rate of proliferation of clonogens is reduced by this agent. It appears that P-30 Protein induces cytostatic effects via modulation of cell transition to quiescence or differentiation. the mechanism of its cytotoxic activity is unclear.