VA13 Cell Lines Research Articles

4+2]-Cycloaddition to 5-Methylidene-Hydantoins and 5-Methylidene-2-Thiohydantoins in the Synthesis of Spiro-2-Chalcogenimidazolones.

Novel hydantion and thiohydantoin-based spiro-compounds were prepared via theDiels-Alder reactions between 5-methylidene-hydantoins or 5-methylidene-2-thiohydantoins and 1,3-dienes (cyclopentadiene, cyclohexadiene, 2,3-dimethylbutadiene, isoprene). It was shown that the cycloaddition reactions proceed regioselectively and stereoselectively with the formation of exo-isomers in the reactions with cyclic dienes andthe less sterically hindered products in the reactions with isoprene. Reactions of methylideneimidazolones with cyclopentadiene proceed viaco-heating the reactants; reactions with cyclohexadiene, 2,3-dimethylbutadiene, and isoprene require catalysis by Lewis acids. It was demonstrated that ZnI2 is an effective catalyst in the Diels-Alder reactions of methylidenethiohydantoins with non-activated dienes. The possibility of alkylation and acylation of the obtained spiro-hydantoinsat the N(1)nitrogen atoms with PhCH2Cl or Boc2O and the alkylation of the spiro-thiohydantoinsat the S atoms with MeI or PhCH2Cl in high yields have been demonstrated. The preparativetransformation of spiro-thiohydantoins into corresponding spiro-hydantoinsin mild conditions by treating with 35% aqueous H2O2 or nitrile oxide has been carried out. The obtained compounds show moderate cytotoxicity in the MTT test on MCF7, A549, HEK293T, and VA13 cell lines. Some of the tested compounds demonstrated some antibacterial effect against Escherichia coli (E. coli) BW25113 DTC-pDualrep2 but were almost inactive against E. coli BW25113 LPTD-pDualrep2.

TERRA R-LOOPS ARE INCREASED IN ACUTE MYELOID LEUKEMIA

Telomeric repeat-containing RNA (TERRA) are long non-coding RNAs transcribed from subtelomeric regions towards telomere ends. TERRA binds to subtelomeric and telomeric regions, forming R-loops, structures composed of DNA-RNA hybrids and displaced single-stranded DNA. TERRA R-loops protect telomeres from DNA damage and promote telomere elongation in short telomeres by enabling telomerase or by activating alternative telomere lengthening. However, high or low TERRA levels are associated with DNA damage and telomere dysfunction. RNaseH1 and RNaseH2 cleave R-loops at telomeres, and regulate R-loops levels. The role of TERRA R-loops in telomere maintenance in human cells is not well understood, since conflicting results derive from experiments using diverse organisms and commercial cell lines, with few studies applying primary human cells. Here we describe for the first time the TERRA physiology in hematopoietic cells of patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), telomeropathies (TEL), and healthy subjects. We initially screened 17 patients with AML, 5 with ALL, 9 with TEL, and 20 healthy subjects. Additionally, 88 AML patients were studied. HeLa, H1, THP1, NB4, and VA13 cell lines were used for functional experiments, and lentivirus vectors containing GFP or RNaseH1-GFP motifs were employed. ATRX, TRF2, TERT, RNaseH1, RNaseH2, TERRA 10q, 15q, 20q, XqYq, XpYp, and 17p expression levels were measured by RT-qPCR. Telomere length was determined by qPCR. R-loops were assessed by immunofluorescence using the S9.6 antibody. Cell line viability was defined by MTT assay. NGS panel was designed to evaluate variants in splicing factors and epigenetic regulators. In the screening population, TERRA expression (10q, 15q, 20q, and XqYq) was significantly increased in AML samples (p < 0.05) in comparison to healthy controls and VA-13 cells. Telomeres were very short in AML samples and TEL patients compared to controls. R-loop formation was significantly increased in AML samples compared to controls (p < 0.01). To examine the TERRA physiology in AML, we further eveluated 88 AML samples. Hierarchical cluster algorithm based on TERRA expression defined two AML groups, one with high and the other with low TERRA expression. Both groups dysplayed short telomeres, but the high TERRA AML group exhibited low TERT and RNaseH2 expression levels (p < 0.05). There was no difference between groups on somatic variants or RNaseH1, TRF2, ATRX, and TRF2 expression levels. AML cell lines were transfected RNaseH1-GFP-containing or empty vector lentiviruses and treated with cytarabine. AML cells with exogenous RNAseH1 expression showed more sensitivity to cytarabine in MTT assays (p < 0.05) than cells transfected with the empty vector. These results show for the first time that TERRA is dysregulated in AML. High TERRA levels are unique to a subset of AML cases with low TERT expression. High TERRA correlated with the accumulation of R-loops, which may be therapeutic targets. TERRA upregulation in AML due to short telomeres is unlikely, given the low TERRA levels in TEL patients. TERRA upregulation did not appear to be directly related to neoplastic differentiation, given low TERRA in ALL. TERRA expression and R-loops are dysregulated in AML. TERRA R-loops inhibition increases chemotherapy-induced apoptosis in AML cell lines and may serve as a therapeutic target.

Syntheses and structure-activity relationship study of santamarine, douglanine, dihydrosantamarine and four series of 1-/3-oxygenated eudesman-12,6α-olides

Herein, we report the syntheses and structure-activity relationships of Santamarine, Douglanine, dihydrosantamarine, 1-oxygenated and 3-oxygenated eudesman-12,6α-lactones. The anti-inflammatory and anti-cancer activities were measured by the inhibition on induction of intercellular adhesion molecule (ICAM-1), killing function of cytotoxic T lymphocytes (CTLs), and cytotoxic activities to cell lines (VA-13, HepG-2, and WI-38), respectively. α-Methylene-γ-lactones 13 with a 1-oxo-2-en moiety showed significant inhibition toward ICAM-1(IC50 12.8 μM), the killing function of CTLs (IC50 11.0 μM) and the VA-13 cell line (IC50 2.84 μM), 21 with α-bromo-ketone moiety showed significant inhibition to VA-13(IC50 1.85 μM). Both 13 ((cell viability)/(inhibitory activity of ICAM-1) = 6.0-fold, WI-38/VA-13 = 1.28-fold) and 21 (WI-38/VA-13 = 1.28-fold) were within safety satisfaction. To conclude, the α,β-unsaturated-ketone and α-bromo-ketone moieties were essential for α-methylene-eudesman-12,6α-olides' anti-inflammatory and anti-cancer activities.

Dispirooxindole-β-Lactams: Synthesis via Staudinger Ketene-Imine Cycloaddition and Biological Evaluation.

In this work, we present the first synthesis of dispirooxindole-β-lactams employing optimized methodology of one-pot Staudinger ketene-imine cycloaddition with N-aryl-2-oxo-pyrrolidine-3-carboxylic acids as the ketene source. Spiroconjugation of indoline-2-one with β-lactams ring is considered to be able to provide stabilization and wide scope of functionalization to resulting scaffolds. The dispipooxindoles obtained demonstrated medium cytotoxicity in the MTT test on A549, MCF7, HEK293, and VA13 cell lines, and one of the compounds demonstrated antibacterial activity against E. coli strain LPTD.

Nitrosylsulfuric acid in the synthesis of 5-chloroisoxazoles from 1,1-dichlorocyclopropanes

Nitrosylsulfuric acid is shown to be a usable reagent for the synthesis of 5-chloroisoxazoles from readily available 1,1-di-chlorocyclopropanes via nitrosation–heterocyclization reaction. A cytotoxicity of some of the prepared 5-chloroisoxazoles towards MCF7, A549, HEK293T and VA13 cell lines was evaluated.

Synthesis of dispirooxindoles containing N-unsubstituted heterocyclic moieties and study of their anticancer activity

A convenient method is proposed for the synthesis of N-unsubstituted spiroxindoles with different heterocyclic moieties (2-thiohydantoin, hydantoin, and thiazolidine) by the regio-selective 1,3-dipolar cycloaddition of azomethine ylides, generated from isatins and sarcosine, to arylidene derivatives of corresponding heterocycles. The cytotoxicity of compounds was tested by the MTT method against MCF7, A549, HEK, and VA13 cell lines and compared with the anticancer drug Nutlin-3a. The best bioactivity was observed for hydantoin-based dispiroindolinones, the most cytotoxic compound demonstrated selectivity against A549 lung cancer cells with an IC50 value of 6.6±1.6 μmol L−1.

Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts

Nonmuscle myosin II (NMII) plays an important role in cytokinesis by constricting a contractile ring. However, it is poorly understood how NMII isoforms contribute to cytokinesis in mammalian cells. Here, we investigated the roles of the two major NMII isoforms, NMIIA and NMIIB, in cytokinesis using a WI-38 VA13 cell line (human immortalized fibroblast). In this cell line, NMIIB tended to localize to the contractile ring more than NMIIA. The expression level of NMIIA affected the localization of NMIIB. Most NMIIB accumulated at the cleavage furrow in NMIIA-knockout (KO) cells, and most NMIIA was displaced from this location in exogenous NMIIB-expressing cells, indicating that NMIIB preferentially localizes to the contractile ring. Specific KO of each isoform elicited opposite effects. The rate of furrow ingression was decreased and increased in NMIIA-KO and NMIIB-KO cells, respectively. Meanwhile, the length of NMII-filament stacks in the contractile ring was increased and decreased in NMIIA-KO and NMIIB-KO cells, respectively. Moreover, NMIIA helped to maintain cortical stiffness during cytokinesis. These findings suggest that appropriate ratio of NMIIA and NMIIB in the contractile ring is important for proper cytokinesis in specific cell types. In addition, two-photon excitation spinning-disk confocal microscopy enabled us to image constriction of the contractile ring in live cells in a three-dimensional manner.

Synthesis and cytotoxicity of novel dispiro derivatives of 5-arylidenoxazolones, potential inhibitors of p53—MDM2 protein-protein interaction

Regioselective synthesis of new dispiro indolinones combining both an indolinone and an oxazolone fragment in their structure comprised the 1,3-dipolar cycloaddition of azomethine ylides, generated in situ from isatin and sarcosine, at 2-aryl-5-arylmethylidene-substituted 1,3-oxazol-5(4H)-ones. When ortho and para halogen atoms were present in the aromatic substituents of the starting oxazolones, complex mixtures containing large amounts of oxazoline ring opening products and their dispiro derivatives were formed. The cytotoxicity of compounds was tested by MTT on LNCaP, PC3, HCT116, MCF7, A549, HEK, and VA13 cell lines. The compound possessing the best cytotoxicity revealed the IC50 = 1.08±0.96 μM towards the p53- expressing LNCaP cells and lower activity (IC50 = 3.21±1.45 μM) towards the non-expressing p53 protein PC3 cells, however, it has proved inactive towards the HCT cells, both expressing (HCТ+/+) and non-expressing (HCT–/–) p53.

Telomere Biology Gene Mutation and Transplant Outcome in Patients with Dyskeratosis Congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome known as the prototype of telomere diseases. In addition to the clinical triad (nail dystrophy, hyperpigmentation, and leukoplakia), very short telomeres (below the 1st percentile) is a marker for the diagnosis of DC (Calado & Young, NEJM 2009). Telomere dysfunction was associated with DC after discovery of DKC1 mutations in patients presenting the X-linked form of the syndrome. Novel mutations in telomere biology genes (TERC, TERT, NOP10, NHP2, TINF2, TCAB1, CTC1, RTEL1, ACD, and PARN) have been described in patients with DC. Variations in all these genes can affect telomere protection and maintenance, leading to telomere shortening and development of telomeropathies. In this study, we mapped the TERT, TERC, DKC1, and TINF2 genes for mutations in 15 patients (median age = 10 years; M/F = 11/4) with DC and very short telomeres, in order to classify these cases in a molecular level and determine the frequency of these mutations in our cohort. Survival for twelve patients who underwent allogeneic hematopoietic stem cell transplant (HSCT) was assessed and correlated with mutational status and telomere length. Diagnosis of DC was made according to the definition of Calado & Young (2009). Telomere length was measured in nucleated blood cells by flow-FISH and mutational screening was performed on genomic DNA extracted from peripheral blood cells by direct sequencing. Seven non-synonymous mutations were identified in TINF2 (40%), two in DKC1 (13%), one in TERT (6%), and one in TERC (6%). The TINF2 variants R282H and R282C had been already described as pathogenic, as well T66A and A353V DKC1 variants (Knight et al, 1999; Savage et al, 2008; Walne et al, 2008). The heterozygous variant R282H (c. 845 G>A) in TINF2 were found in 4 unrelated patients. One of them also harbor the variants Q120R and Q157H in the same gene. The heterozygous mutation in TINF2 R282C (c. 844 C>T) was found in one patient, that also presented the common polymorphism A279T in TERT. The pathogenic variants T66A (c.196A>G) and A353V (c.1058C>T) in DKC1 were found in two different male patients. Moreover, three novel mutations were identified in our cohort, r.94 C>T in TERC, F290C in TINF2, and R696Cin TERT. The heterozygous mutation r.94 (C>T) found in TERC was located at the pseudoknot P2b region of the gene and the patient who carries that presented a severe aplastic anemia and all DC clinical triad. The novel heterozygous F290C (c.859 T>G) variant is located at the "hot spot" in exon 6 of TINF2 andwas found in one patient that presented a severe phenotype of DC. In silico analysis with SIFT and Polyphen-2 predicted that this variant is not tolerated and probably damaging, which is consistent with the pathogenicity of the mutation. The homozygous mutation R696C (c.2086 C>T) in TERT was found in one patient and also in his two brothers. All of them presented reduced blood cell count, clinical features of DC, and severe aplastic anemia. The family screening identified the father and sister as heterozygous for the same mutation, but both asymptomatic. DNA sample from the mother were not available for this study. In silico analysis by SIFT and Polyphen 2.0, predicted that the R696C mutation is not tolerant and possible damaging to telomerase activity, respectively. To validate in silico analysis, TRAP assay with cell lysates obtained from telomerase-negative VA13 cell line transfected with wild type or R696C mutated TERT vector and TERC vector is under evaluation. Consistently with previously studies, telomere length in patients with TINF2 mutations were the shortest compared with the other telomeres genes mapped in this study. Although, the phenotype and severity of the disease does not appear to change according to the mutated gene. Also, the mutational status (p=0.28) or telomere length (p=0.21) did not influence the survival rates of patients after HSCT.Flow-FISH was able to identify patients with very short telomeres and validated telomere length measurement as a diagnostic tool for DC. Direct sequencing of the most commonly mutated genes in DC in a cohort of patients with telomeres below 1st percentile was able to characterize the genetic cause of this disease in more than 70% of the cases. The identification of genetic defect in DC can manage clinical decisions and is essential to genetic counseling prior to bone marrow transplantation. DisclosuresNo relevant conflicts of interest to declare.

A Large Mennonite Family with a Novel K570N TERT Gene Mutation: Association with a Clinical Spectrum of Bone Marrow Failure, Acute Myeloid Leukemia, and Acute Liver Failure.

Mutations in telomere repair complex genes TERT (encoding telomerase reverse transcriptase) and TERC (telomerase RNA component) are associated with bone marrow failure, especially acquired aplastic anemia and dyskeratosis congenita. Low telomerase activity leads to short telomeres of leukocytes, predisposing highly proliferative tissues such as the bone marrow to early senescence and exhaustion of the stem cell compartment. Telomere repair gene mutations have been suggested to result in disease anticipation, defined as earlier and/or worsening clinical manifestations in successive generations. We have identified a six-generation pedigree in a large Mennonite family carrying a novel TERT mutation (K570N), which localizes in the catalytic domain with reverse transcriptase activity (RT domain). The index patient is a 26 year old male dairy farmer with a ten-year history of severe aplastic anemia (5% bone marrow cellularity) unresponsive to immunosuppression. A long history of hematologic diseases was well known and named in the family--the patient's paternal great-great-grandmother had died of a severe blood disorder at age 65 years. However, the great-grandmother and the grandfather had never presented any hematological disease. The patient's father had myelodysplastic syndrome at age 33 years, evolving to acute myeloid leukemia and death due to failure to recover blood counts after chemotherapy. One of the proband's paternal aunts had aplastic anemia develop when she was a young woman and has been transfusion-independent for decades in response to chronic androgen therapy. A second proband's paternal aunt underwent a liver transplant at age 20 for submassive hepatic necrosis with fibrosis. A third proband's paternal aunt has macrocytosis only at age 47. Two sisters (ages 21 and 23) also have macrocytosis in the absence of other hematological abnormality, and two other sisters are healthy. Genetic analysis showed that TERT K570N mutation is present in the patient's paternal (including grandfather, three aunts and two affected sisters) but not maternal relatives (making his father an obligatory carrier). The patient's oldest of three sons, now age four years, has the mutation but is asymptomatic. There was no nail dystrophy, leukoplakia or skin hyperpigmentation in any of the TERT K570N carriers; although the index patient and some of his relatives showed early graying of hair, this characteristic did not track with the mutation. Telomere shortening of leukocytes, as measured by Flow-FISH, tracked to the mutation in three generations analyzed, being shortest in the proband and in his aunt with marrow failure. Mutagenized TERT vectors transfected into telomerase-deficient VA13 cell lines yielded no telomerase activity using the telomeric repeat amplification protocol (TRAP) assay, whereas when wild-type TERT vectors were co-transfected, telomerase activity was approximately half of wild-type transfected only, indicating haploinsufficiency as a mechanism of telomere shortening. Our results confirm the association between aplastic anemia and TERT mutations. The pattern of hematologic disease in this kindred does not support disease anticipation in TERT mutations. Most remarkably, there is a likely relationship between a telomerase gene mutation and hematological malignancy and severe liver disease.

IκBα (inhibitory κBα) identified as labile repressor of MnSOD (manganese superoxide dismutase) expression

Cytokines, phorbol esters, radiation and chemotherapeutic drugs up-regulate the expression of MnSOD (manganese superoxide dismutase). Using the VA-13 cell line, we studied the regulation of SOD2 upon treatment with PMA. Pre-treatment with CHX (cycloheximide) followed by PMA led to significantly higher levels of MnSOD mRNA compared with those with either agent alone, suggesting de novo synthesis of an inhibitory protein. PMA treatment modulates redox-sensitive transcription factors, therefore we evaluated the effects of this combination treatment upon AP-1 (activator protein 1) and NF-kappaB (nuclear factor kappaB), two trans-acting factors suggested to play a role in SOD2 regulation. Co-administration of CHX and PMA led to a time-dependent increase in the binding activity of NF-kappaB. Therefore we evaluated IkappaBalpha (inhibitory kappaBalpha) and found that co-administration decreased its steady-state level compared with either agent alone, suggesting that enhanced NF-kappaB activation is due to inhibition of IkappaBalpha synthesis. PMA activates PKC (protein kinase C) enzymes which phosphorylate IkappaBalpha, leading to its degradation, therefore we used GF109203X to inhibit PKC activity. Stable transfection utilizing a PMA-responsive element in the human SOD2 gene, showed a concentration-dependent decrease in luciferase and NF-kappaB-binding activity with GF109203X. Western blot analysis indicated the presence of several PKC isoforms in the VA-13 cell line; however, PMA pre-treatment specifically down-regulated alpha and betaI, suggesting a role for one or more of these proteins in SOD2 induction. Taken together, these results indicate that the PKC pathway leading to SOD2 induction proceeds at least in part through NF-kappaB and that inhibition of IkappaBalpha synthesis might serve as a potential pharmacological approach to up-regulate MnSOD.

Elevated superoxide production by active H-ras enhances human lung WI-38VA-13 cell proliferation, migration and resistance to TNF-alpha.

Accumulating evidence has suggested that cellular production of superoxide acts as an intracellular messenger to regulate gene expression and modulate cellular activities. In this report, we set out to investigate the role of active H-ras-mediated superoxide production on tumor cell malignancy in a SV-40 transformed human lung WI-38 VA-13 cell line. Stable transfection and expression of constitutively active mutant V12-H-ras (V12-H-ras) dramatically increased intracellular production of superoxide. The expression of V12-H-ras significantly enhanced cell proliferation, migration and resistance to TNF-alpha treatment compared to that of parental and vector control cells, while expression of wild type H-ras (WT-H-ras) only had modest effects. Upon scavenging by superoxide dismutase and other molecules that decrease the intracellular level of active H-ras mediated superoxide production, cell proliferation, migration and resistance to TNF-alpha were significantly reduced. Furthermore, we demonstrated that the activation of membrane NADPH oxidase activity by expression of active H-ras contributed to the intracellular superoxide production. The causal relationship between membrane superoxide production and increased cell proliferation, migration, and resistance to TNF-alpha by the expression of active H-ras, has provided direct evidence to demonstrate that superoxide acts as an intracellular messenger to cascade ras oncogenic signal relay and to modulate tumor malignant activity.

Functional roles of E2F in cell cycle regulation.

The E2F family of transcription factors play a key role in G1-S progression. A dominant negative mutant (E2F97) of E2F1 containing the DNA binding domain of E2F1 under the control of a tetracycline-responsive promoter was constructed. Stable transfectants were produced in the pRb-lacking SaOS-2 cell line and SV40-transformed VA-13 cell line, respectively. Induction of E2F97 by tetracycline withdrawal resulted in strong inhibition of the E2F transcriptional activity and a decreased percentage of cells in S-phase. To understand the mechanism(s) by which E2F97 exerts its effect on the cell cycle, the effect of E2F97 on expression of various cell cycle proteins was examined. Upon induction of E2F97, a significant decrease in the levels of both dihydrofolate reductase and thymidylate synthase was observed in transfectants derived from both cell lines. Induction of E2F97 also led to a decrease in cyclin A and D1 protein levels. Regulation of cyclin A by E2F97 occurred at the transcriptional level. In addition, in VA13 cells, induction of E2F97 resulted in down-regulation of the tumor suppressor protein p53. These data suggest that E2F regulates both G1 and S-phase cyclins and that there may be a potential positive feedback regulatory loop between E2F and cyclin D1.

Fractal motion of mammalian cells

Hurst's rescaled range analysis has been applied to electrical signals that reflect the motion of fibroblastic human cells. According to the analysis the cells have a fractional Brownian motion with a Hurst exponent of about H=0.65. The normal WI-38 cell line has a Hurst exponent that is slightly smaller than the transformed WI-38 VA13 cell line.

DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells

Mitomycin C (MMC), a quinone-containing antitumor drug, has been shown to alkylate DNA and to form DNA cross-links. The ability of MMC to alkylate O 6-guanine and to form interstrand cross-links (ISC) has been studied using Mer + and Mer − human embryonic cells. Mer + (IMR-90) cells have been reported to contain an O 6-alkylguanine transferase enzyme and are, in general, more resistant to alkylating agents than the Mer − (VA-13) cell line, which is deficient in the repair of O 6-lesions in DNA. Studies reported here show that MMC is more cytotoxic to VA-13 cells compared to IMR-90 cells. The alkaline elution technique was used to quantify MMC-induced ISC, and double strand breaks (DSB) in these cells. The drug-dependent formation of DSB was significantly lower in IMR-90 cells than in VA-13 cells. In contrast, no significant difference in cross-linking could be detected at the end of 2-h drug treatment. Although a small increase in cross-link frequency was observed in the VA-13 cell line relative to the IMR-90 cell line 6 h post drug treatment, it is not clear whether monoalkylated adducts at the O 6-position are formed, and contribute to cross-link formation for differential cytotoxicity in VA-13 cells. Electron spin resonance and spin-trapping technique were used to detect the formation of hydroxyl radical from MMC-treated cells. Our studies show that MMC significantly stimulated the formation of hydroxyl radical in VA-13 cells, but not in the IMR-90 cells. The formation of the hydroxyl radical was inhibited by superoxide dismutase (SOD) and catalase. In addition, the presence of these enzymes partially protected VA-13 cells from MMC toxicity but not IMR-90 cells. Further studies indicated that the decreased free radical formation and resistance to MMC may be due to the increased activities of catalase and glutathione transferase in the IMR-90 cell line. These results suggest that MMC-dependent DNA damage (alkylation and DNA DSB) and the stimulation of oxy-radical formation may play critical roles in the determination of MMC-induced cell killing.

Pneumocystis carinii: Growth variables and estimates in the A549 and WI-38 VA13 human cell lines

Recent studies indicate that rat Pneumocystis carinii can be propagated in the A549 cell line, an alveolar epithelioid cell line derived from human lung carcinoma. In the present study, growth of P. carinii was compared in the A549 cell line and the WI-38 VA13 subline 2RA, an SV40 transformed derivative of the human fetal fibroblast cell line with epithelioid morphology. Similar P. carinii growth occurred in both cell lines under optimal conditions, but the WI-38 VA13 cell line was usually more sensitive to changes in the culture system. Growth of P. carinii was affected by temperature, environmental gas mixture, motion of the cultures, and source and concentration of serum additives, but not by the presence of antibodies in the medium. A technique was developed for quantitating P. carinii in the lung inoculum which permitted analysis of P. carinii growth during the first 24 hr of culture. Inverted microscope and oil immersion phase-contrast microscopy were very helpful in monitoring the organism's stages of development and viability. Thus, this culture system should be helpful in establishing standard methodology for in vitro work with P. carinii.

Serological Detection of Common Human Melanoma Membrane Antigens by Microcomplement Fixation and Immunofluorescence

An antiserum to human melanoma antigens was obtained from a melanoma patient after immunization with autologous irradiated cultured melanoma cells and bacillus Calmette-Guerin. Using the microcomplement fixation assay, the antiserum, at a titer of 1/1,800, was noted to bind strongly with 7 of 10 allogeneic cultured human melanoma cells lines. However, using the indirect immunofluorescence test and serum at a much lower titer (1/8), only 3 of the 10 melanoma cell lines were positive. Using both microcomplement fixation and indirect immunofluorescence, no significant reactivity was noted in several nonmelanoma cell lines including Hela, human lung adenocarcinoma, human prostatic carcinoma, WI-38 and VA-13 cell lines. These data suggest that common melanoma membrane antigens exist on 7 of 10 cultured human melanoma cell lines as tested by microcomplement fixation and that this assay is more sensitive than immunofluorescence. These common melanoma membrane antigens may eventually be extracted, purified and used for specific immunodiagnosis and immunotherapy.