A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells.

The polypeptides synthesized during the cell cycle of HeLa cells were analyzed by means of two-dimensional gel electrophoresis followed by fluorography under conditions in which the position of 700 polypeptides (acidic and basic) could be reproducibly assessed. Mitotic cells obtained by mechanical detachment and synchronized cells in other stages of the cell cycle were labeled with [35S]methionine for 30-min pulses or for long terms starting at the beginning of each phase. Visual comparison of the polypeptide maps obtained in the different stages of the cell cycle showed that these were strikingly similar, and there was no indication that the synthesis of any of the detected polypeptides was confined to only one of the cell cycle phases. Quantitation of 99 abundant polypeptides (acidic and basic) in pulse-labeled and long-term labeled cells revealed that the relative amount (i.e., the rate of synthesis) of most polypeptides, including total actin, alpha-actinin, 6 abundant basic nonhistone proteins, and 13 major acidic proteins present in Triton cytoskeletons, remains constant throughout the cell cycle. Among the few variable polypeptides (markers), we have identified alpha- and beta-tubulin (increase in M), the subunit of the 100-A filament protein "fibroblast type" (decreases in M), and a 36,000 mol wt acidic cytoarchitectural protein that increases in S. A few other unidentified polypeptides have also been found to vary in M and in M and G2, but no marker was found in G1.

Monoclonal antibody specific for human nuclear proteins IEF 8Z30 and 8Z31 accumulates in the nucleus a few hours after cytoplasmic microinjection of cells expressing these proteins.

A monoclonal antibody (mAB 1C4C10) that reacts specifically with human nuclear proteins IEF 8Z30 and 8Z31 (charge variants; HeLa protein catalogue number; Bravo, R., and J. E. Celis, 1982, Clin. Chem., 28:766-781) has been microinjected into the cytoplasm of cultured cells that either express (primates) or lack these proteins (at least having similar molecular weights and pIs; other species), and its cellular localization has been determined by indirect immunofluorescence. Nuclear localization (nucleolar and nucleoplasmic) of the antibody was observed only in cells expressing these antigens, suggesting that a determinant present in IEF 8Z30 and 8Z31 is required for cytoplasm-nuclear translocation. Nuclear migration was not inhibited by cycloheximide, implying that these proteins may shuttle between nucleus and cytoplasm. The results assumed to support the signal rather than the free diffusion model are further supported by microinjection experiments using antibodies (proliferating cell nuclear antigen/cyclin, DNA) that react with nuclear components but do not recognize cytoplasmic antigens. Furthermore, they raise the possibility that some nonnuclear proteins may be transported to the nucleus by interacting with proteins harboring nuclear location signals.

Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis.

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.

Proteins IEF (isoelectric focusing) 31 and IEF 46 are keratin-type components of the intermediate-sized filaments: keratins of various human cultured epithelial cells.

Mouse polyclonal antibodies have been raised against two human proteins (IEF [isoelectric focusing] 31, Mr = 50,000; IEF 46, Mr = 43,500) that have previously been shown to be present in HeLa cytoskeletons enriched in intermediate-sized filaments. Immunoprecipitation studies show that both proteins share common antigenic determinants with each other and with the putative human keratins IEF 36 and 44, also present in HeLa cytoskeletons. Indirect immunofluorescence studies showed that both antibodies revealed similar filamentous networks in various cultured epithelial cells of human origin. These included AMA (transformed amnion), HeLa (cervical carcinoma), normal amnion cells, Fl-amnion (transformed amnion), WISH-amnion (transformed amnion), Chang liver (liver), and Detroid-98 (sternal marrow). Human cells that did not react with both antibodies included skin fibroblasts, lung fibroblasts (WI-38), SV40-transformed lung fibroblasts, Molt 4 (leukemia), lymphocytes, and monocytes. These results were in complete agreement with the presence or absence of both proteins in two-dimensional gels of the different cell types. Exposure of AMA cells to demecolcine (24 h; 10 micrograms/ml) caused the total collapse of vimentin filaments but, as seen by indirect immunofluorescence, caused only a partial redistribution of the IEF 31 and 46 filaments. These results are taken to suggest that both proteins are components of the intermediate-sized filaments of the "keratin" type. The antibodies could be clearly differentiated by staining human bladder carcinoma EJ 19 cells, as only the IEF 46 antibody stained a filamentous network in these cells The occurrence of keratins IEF 31, 36, 44, and 46 in different cultured human epithelial cells has been studied using two-dimensional gel electrophoresis.

Neurological disturbances, premature lethality, and central myelination deficiency in transgenic mice overexpressing the homeo domain transcription factor Oct-6.

Pit, Oct, Unc (POU) homeo domain transcription factors have been implicated in various developmental processes, including cell division, differentiation, specification, and survival of specific cell types. Although expression of the transcription factor Oct-6 in oligodendroglia is confined to the promyelin stage and is downregulated at the myelin stage of development, the effect of Oct-6 overexpression on oligodendrocyte development has not been established. Here we show that transgenic animals overexpressing Oct-6 at late oligodendrocyte development develop a severe neurologic syndrome characterized by action tremors, recurrent seizures, and premature death. Axons in the central nervous system of Oct-6 transgenics were hypomyelinated, hypermyelinated, or dysmyelinated, and ultrastructural analyses suggested that myelin formation was premature. The vulnerability of developing oligodendroglia to Oct-6 deregulation provides evidence that the POU factor may play a direct role in myelin disease pathogenesis in the mammalian CNS.

EF-hands at atomic resolution: the structure of human psoriasin (S100A7) solved by MAD phasing.

BACKGROUND: The S100 family consists of small acidic proteins, belonging to the EF-hand class of calcium-binding proteins. They are primarily regulatory proteins, involved in cell growth, cell structure regulation and signal transduction. Psoriasin (S100A7) is an 11.7 kDa protein that is highly upregulated in the epidermis of patients suffering from the chronic skin disease psoriasis. Although its exact function is not known, psoriasin is believed to participate in the biochemical response which follows transient changes in the cellular Ca2+ concentration. RESULTS: The three-dimensional structure of holmium-substituted psoriasin has been determined by multiple anomalous wavelength dispersion (MAD) phasing and refined to atomic resolution (1.05 A). The structure represents the most accurately determined structure of a calcium-binding protein. Although the overall structure of psoriasin is similar to those of other S100 proteins, several important differences exist, mainly in the N-terminal EF-hand motif that contains a distorted loop and lacks a crucial calcium-binding residue. It is these minor differences that may account for the different specificities among members of this family. CONCLUSIONS: The structure of human psoriasin reveals that this protein, in contrast to other S100 proteins with known structure, is not likely to strongly bind more than one calcium ion per monomer. The present study contradicts the idea that calcium binding induces large changes in conformation, as suggested by previously determined structures of apo forms of S100 proteins. The substitution of Ca2+ ions in EF-hands by lanthanide ions may provide a general vehicle for structure determination of S100 proteins by means of MAD phasing.

Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas.

Multifocal recurrent papillary tumors provide a unique model system to study the molecular mechanisms underlying the steps involved in transitional cell carcinoma progression and offer a valuable source of material to search for biomarkers that may form the basis for diagnosis, prognosis, and treatment. We have examined the protein expression profiles of normal bladder urothelium and of 63 transitional cell carcinomas of various histopathological grades and T stages using high-resolution, two-dimensional gel electrophoresis, microsequencing, mass spectrometry, and a two-dimensional gel protein database approach for polypeptide identification (http://biobase.dk/cgi-bin/celis). In general, the results revealed a striking similarity between the overall qualitative expression patterns of papillary tumors of all grades, as well as of papillary and solid tumors of grade III. With few exceptions, tumors of grades I-III expressed, albeit at different levels, all of the keratins (7, 8, 13, 17, 18, 19, and 20) found in the normal urothelium. Grade IV tumors lacked or expressed reduced levels of keratin 13 but most resembled low-grade tumors. One invasive grade IV tumor, however, expressed a fibroblast-like protein phenotype. Four proteins that were expressed by normal urothelium and were lost at various stages of progression were identified as glutathione S-transferase mu, prostaglandin dehydrogenase (PGDH), a fatty acid binding protein with homology to the adipocyte isoform (A-FABP), and keratin 13. The percentage of tumors expressing A-FABP was very high in low-grade lesions but decreased drastically (P = 0.0006) in grade III and IV neoplasms. In addition, low-grade tumors contained more A-FABP than their high-grade counterparts. The stage of the disease was also statistically (P = 0.0269) related to the presence or absence of A-FABP in grade III tumors. Similar analysis of glutathione S-transferase mu and PGDH showed a statistically significant decrease of these proteins in high-grade (grades III and IV) tumors (P = 0.0026 and P = 0.0044, respectively). Only PGDH showed a suggestive correlation (P = 0.0775) with the stage of the disease in grade III tumors. Keratin 13 showed a drastic decrease in grade IV tumors. In addition to identifying biomarkers that may have prognostic value, our studies have suggested that A-FABP is an important component of the pathway(s) leading to bladder cancer development.

Proteomics and immunohistochemistry define some of the steps involved in the squamous differentiation of the bladder transitional epithelium: a novel strategy for identifying metaplastic lesions.

Here, we present a novel strategy for dissecting some of the steps involved in the squamous differentiation of the bladder urothelium leading to squamous cell carcinomas (SCCs). First, we used proteomic technologies and databases (http://biobase.dk/cgi-bin/celis) to reveal proteins that were expressed specifically by fresh normal urothelium and three SCCs showing no urothelial components. Thereafter, antibodies against some of the differentially expressed proteins as well as a few known keratinocyte markers were used to stain serial cryostat sections (immunowalking) of biopsies obtained from bladder cystectomies of two of the SCC-bearing patients (884-1 and 864-1). Because bladder cancer is a field disease, we surmised that the urothelium of these patients may exhibit a spectrum of abnormalities ranging from early metaplastic stages to invasive disease. Immunohistochemical analysis revealed three types of non-keratinizing metaplastic lesions (types 1-3) that did not express keratins 7, 8, 18, and 20 (expressed by normal urothelium) and could be distinguished based on their staining with keratin 19 antibodies. Type 1 lesions showed staining of all cell layers in the epithelium (with differences in the staining intensity of the basal compartment), whereas type 2 lesions exhibited mainly basal cell staining. Type 3 lesions did not stain with keratin 19 antibodies. In cystectomy 884-1, type 3 lesions exhibited the same immunophenotype as the SCC and may be regarded as precursors to the tumor. Basal cells in these lesions did not express keratin 13, suggesting that the tumor, which was also keratin 13 negative, may have arisen from the expansion of these cells. Similar results were observed with cystectomy 864-1, which showed carcinoma in situ of the SCC type. SCC 864-1 exhibited both keratin 19-negative and -positive cells, implying that the tumor arose from the expansion of the basal cell compartment of type 2 and 3 lesions. Besides providing with a novel strategy for revealing metaplastic lesions, our studies have shown that it is feasible to apply powerful proteomic technologies to the analysis of complex biological samples under conditions that are as close as possible to the in vivo situation.

Proteome profiling of bladder squamous cell carcinomas: identification of markers that define their degree of differentiation.

One hundred fifty fresh bladder tumors were analyzed blindly by two-dimensional PAGE in combination with proteome identification techniques (microsequencing and mass spectrometry) and immunofluorescence of cryostat sections. Of these, six showed protein expression patterns corresponding to squamous cell carcinomas (SCCs). All tumors were already invasive at the time of presentation, and in most cases, the histopathological grade could not be determined with certainty. The more differentiated of the tumors included SCC 589-1, a lesion showing extensive keratinization, and 536-1, a pure SCC that resembled normal skin growing invasively into the muscle. Both tumors expressed keratins 5, 6, 10, 14, 16, 17, and 20, as well as the differentiation-associated proteins psoriasin, psoriasis-associated fatty acid-binding protein (PA-FABP), and galectin 7. SCC 589-1, however, exhibited higher levels of keratin 10, PA-FABP, and galectin 7 and, in addition, expressed keratins 13, 15, and 19, which were not detected in the pure SCC. Involucrin, glutathione S-transferase pi, stratifin (14-3-3 sigma), and the SCC antigen 1, on the other hand, were less abundant in SCC 589-1. In comparison, less-differentiated tumors did not express keratin 10 and were characterized by a decreased expression of keratin 14, psoriasin, PA-FABP, galectin 7, and stratifin (14-3-3 sigma). Indeed, two of these lesions (553-1 and 651-1) could be readily lined up in order of decreasing degree of differentiation based on the expression of these markers. The degree of differentiation of the other two SCCs could not be assessed with certainty because they may represent special cases (SCC 646-1, solid tumor; SCC 485-1, special differentiation pattern). All six SCCs externalized psoriasin to the urine, supporting the contention that this protein, alone or in combination with other polypeptides, may represent a useful marker for the early detection of these lesions.

Failure of central nervous system myelination in MBP/c-myc transgenic mice: evidence for c-myc cytotoxicity.

c-myc is a member of the helix-loop-helix/leucine zipper family of proteins that modulate the transcriptional activity of specific target genes. Although aberrant c-myc expression has been reported to play a role in multistage carcinogenesis in astrocytic gliomas, little is known about the effects of the expression of c-myc on oligodendrocytes. Using transgenic animals expressing a human c-myc oncogene under transcriptional control of the myelin basic protein gene, we investigated the effect of overexpression of this oncogene in oligodendrocytes. The MBP/c-myc transgenic mice developed severe neurological disturbances characterized by action tremors and recurrent seizures, and premature death during postnatal weeks three to five. Affected transgenic mice of various strains had severely hypomyelinated central nervous systems and expressed low levels of c-myc, myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs in the brain. These c-myc transgenic mice also exhibited an increased number of TUNEL positive nuclei, which in most cases were located in cells that expressed c-myc, as judged by double immunohistochemistry. There was no evidence of brain tumors in the c-myc transgenic mice, including heterozygous mice from two strains that had normal lifespans. These observations indicate that the myelin deficiency observed in the MBP/c-myc transgenic animals results from a cytotoxic effect of the c-myc transgene.

Identification, molecular cloning, expression and chromosome mapping of a family of transformation upregulated hnRNP-K proteins derived by alternative splicing.

Acidic nuclear proteins (M(r) between 64,000 and 66,000; pI 4.9 to 5.5) that are highly upregulated in transformed cells and that belong to the hnRNP-K family have been identified using a monoclonal antibody (mAB B4B6) that distinguish between quiescent and proliferating human keratinocytes. The family, which is composed of four major proteins (hnRNPs-K A, B, C and D) and their modified forms, is present in similar overall levels in quiescent and proliferating normal keratinocytes although clear differences were observed in the levels of some of the individual variants. Immunofluorescence staining of proliferating normal keratinocytes with mAB B4B6 showed that about 40% of the keratinocytes, corresponding mainly to G1 and to half of the cells in S-phase, reacted with the antibody depicting a dotted, nucleoplasmic staining that excluded the nucleolus. Only 3 to 4% of the quiescent keratinocytes reacted with the antibody while simian virus 40 (SV40) transformed keratinocytes (K14) stained constitutively throughout the cell cycle. Using mAB B4B6 as a probe we cloned a cDNA coding for one member of the family (hnRNP-K B) and this was used to screen for additional family members. Sequencing of the positive clones revealed four different cDNAs, all resulting from alternative splicing of a common primary transcript of a gene that mapped to chromosome 9. Expression of the cDNAs in the vaccinia virus system confirmed their identity as hnRNPs-K A, B, C and D and showed that their modified forms are phosphorylated. All four hnRNPs bound poly(rC) on NorthWestern blots, although the more acidic of the phosphorylated forms, did so at a much reduced level. hnRNP-K has been implicated in pre-mRNA metabolism of transcripts containing cytidine-rich sequences and our results point towards a role during cell cycle progression.

Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway.

We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human keratinocyte proteins) that share peptide sequences with each other, with protein 14-3-3 and with the kinase C inhibitory protein. Immunofluorescence staining of keratinocytes showed that two of these proteins (IEF SSPs 9124 and 9126) localize to the Golgi apparatus, while stratifin is distributed diffusely in the cytoplasm. Significant levels of stratifin, and in smaller amount the sample spot proteins 9124, 9125 and 9126, were detected in the medium of cultured human keratinocytes suggesting that they are partially secreted by these cells. Two-dimensional gel analysis of proteins from cultured human cells and fetal tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium. We have cloned and sequenced cDNAs coding for members of this family. The complete identity of the sequenced peptides from stratifin with the amino acid sequence translated from the stratifin cDNA clone indicated that this cDNA codes for stratifin. The identity of clones 1054, HS1 and AS1 is less clear as, with few exceptions, none of the individual peptide sequences fits the predicted protein sequences. The polypeptides synthesized by clones 1054 and HS1 in the vaccinia expression system, on the other hand, comigrate with proteins 9126 and 9124, suggesting cell-type-specific expression of members of the protein family. Database searches indicated that clone HS1 corresponds to a human T-cell cDNA 14-3-3 clone, while the high level of similarity of clones 1054 and AS1 with the 14-3-3 beta and eta sequences respectively, suggested that they code for the human equivalent of the two bovine proteins. Microsequence data indicated that IEF SSP 9124 corresponds to the human homolog of bovine 14-3-3 gamma.

2D protein electrophoresis: can it be perfected?

23 years after O'Farrel developed two-dimensional gel electrophoresis we still debate if the technique can be improved, or if there are other alternative separation technologies that can challenge its central position in proteomic projects. These questions are relevant as the pharmaceutical industry expects proteomic studies to provide novel protein targets for drug discovery and diagnostics. In our opinion, there are various aspects of the technology that can be improved, including resolution, sample preparation and detection, but so far there is no alternative technique(s) available, or any under development, that can replace it.

PCNA (cyclin) autoantibodies and monoclonal antibodies reveal similar patterns of cyclin (PCNA) antigen staining in human cultured cells.

Double immunofluorescence and [3H]thymidine autoradiographic analysis of the same field of transformed human amnion cells (AMA) reacted with proliferating cell nuclear antigen (PCNA) autoantibodies and a monoclonal antibody (mAb 19F4) specific for cyclin (PCNA) revealed similar patterns and sequence of cyclin (PCNA) antigen staining during S-phase. These results suggest that immunofluorescence patterns obtained with PCNA autoantibodies reflect in fact patterns of cyclin (PCNA) antigen staining.

Mid to late S-phase replication of the nucleolus in lymphoid human Molt-4 cells.

Indirect immunofluorescence staining of synchronized lymphoid human Molt-4 cells with proliferating cell nuclear antigen autoantibodies specific for cyclin revealed nucleolar staining only in cells in mid to late S-phase. These results together with similar earlier observations in epithelial and fibroblasts cells indicate that this organelle replicates in mid to late S-phase in cultured somatic cells.

Secreted proteins from normal and SV40 transformed human MRC-5 fibroblasts: toward establishing a database of human secreted proteins.

Analysis by means of computerized two-dimensional gel electrophoresis (NEPHGE, IEF) of the [35S]-methionine labeled proteins secreted by normal human MRC-5 fibroblasts revealed 476 polypeptides (258 acidic and 218 basic), many of which appeared as charge trains due to modification. Similar analysis of the proteins secreted by SV40 transformed MRC-5 fibroblasts (MRC-5 V2) showed a striking decrease in the levels of many of these proteins as well as the appearance (or increased synthesis) of 47 polypeptides that were either absent or present in very low amounts in normal cells. Of the major secreted polypeptides whose relative proportion decreased dramatically in the MRC-5 V2 cells, 15 were found to be abundant components of other normal (nontransformed) fibroblasts (W138, Xeroderma pigmentosum cell lines). Low levels of these radioactively labeled polypeptides were observed in transformed human cell lines of fibroblast (W138, SV40, HT1080), epithelial (HeLa, transformed amnion cells (AMA), A431, A459) and myeloid (HL-60) origin. No major secreted polypeptide from MRC-5 V2 cells was synthesized exclusively by the transformed cell lines.

Identification of two human phosphoproteins (dividin and IEF 59dl) that are first detected late in G1 near the G1/S transition border of the cell cycle.

Two-dimensional gel electrophoretic analysis (NEPHGE, IEF) of the [32P]-orthophosphate-labeled proteins synthesized throughout the cell cycle of transformed human amnion cells (AMA) revealed two phosphoproteins (dividin, Mr = 54,000, pl = 8.4; IEF 59dl, Mr = 27,000, pl = 5.7) that are present mainly in S-phase cells. These proteins are first detected at the end of G1, near the G1/S transition border, and their levels reach a maximum late in S-phase. Together with the previously identified nuclear protein cyclin, these phosphoproteins are likely candidates for proteins that may play a role in the regulation of the onset of DNA synthesis and cell division.

Major proteins induced and down-regulated by interferons in human cultured cells: identification of a unique set of proteins induced by interferon-alpha in epithelial, fibroblast, and lymphoid cells.

In all, 40 major polypeptides ranging in molecular weights from 14.5 to 83 kDa were shown to be induced by IFNs alpha (also by IFN-alpha 2b and beta in a few cases) and gamma in human cultured cells of epithelial (transformed amnion cells (AMA)), fibroblast (proliferating and quiescent MRC-5 fibroblasts), and lymphoid origin (Molt-4). With the exception of a heat shock protein (IEF14 or hs x 70) and two tropomyosins (IEFs 52x and 55), none of these proteins corresponded to polypeptides (proliferation-sensitive or others) previously identified and catalogued by us. IFN-alpha induced the highest number of polypeptides in lymphoid cells, while the response to IFN-gamma was more pronounced in cultured epithelial and fibroblast cells. Several of the polypeptides induced by IFNs alpha and gamma were synthesized (albeit at different rates) by the control untreated cells, and in some cell types such as normal human peripheral blood mononuclear cells many were expressed at high levels. Only IFN-alpha-induced a unique set of proteins (alpha 1, 51 kDa; alpha 2, 15 kDa; alpha 19, 78 kDa; and gamma 10, 83 kDa) in all cultured cell types studied, implying that response to this IFN involves a shared biochemical pathway(s). Both IFN-alpha (also IFN-alpha 2b) and beta induced an identical group of proteins in AMA cells in agreement with the fact that type I IFNs share common receptors. IFNs alpha and gamma induced a few common polypeptides, but only gamma 10 (83 kDa) showed increased synthesis in all cell types exposed to either of these IFNs. A total of 28 major cellular polypeptides were down-regulated by IFNs in the various cell type studied. Different sets of proteins were affected, however, in each system, emphasizing the complexity of the mechanisms underlying the action of these factors. Treatment of synchronized G1 AMA cells with IFNs alpha, beta, or gamma (500 IU/ml, final concentration) did not inhibit their progression from G1 to S-phase as determined by indirect immunofluorescence using PCNA autoantibodies specific for cyclin. These observations were in line with the fact that IFNs did not affect dividin or cyclin(PCNA) synthesis (S-phase specific proteins) at least within the first 17 hr after their addition.

Towards establishing comprehensive databases of cellular proteins from transformed human epithelial amnion cells (AMA) and normal peripheral blood mononuclear cells.

Databases of protein information derived from the analysis of two-dimensional gels have been established from transformed human amnion cells (AMA) and peripheral blood mononuclear cells (PBMCs). A total of 1781 [35S]methionine-labeled AMA proteins (1274 IEF, 537 NEPHGE) and a total of 1311 proteins from PBMC (948 IEF, 363 NEPHGE) were resolved and recorded using computerized (PDQ-SCAN and PDQUEST softwares) two-dimensional gel electrophoresis. AMA and PBMC proteins (total, 454: 301 IEF, 153 NEPHGE) were matched both manually and by the computer. Information entered in the AMA database (in most cases for some major proteins) includes: molecular weight, protein name, HeLa protein catalogue number, mouse protein catalogue number, nuclear proteins, phosphorylated proteins, distribution of proteins in Triton X-100 supernatants and cytoskeletons, proliferation- and transformation-sensitive proteins, cell cycle-specific proteins, mitochondrial proteins, proteins matched in normal human embryonal lung MRC-5 fibroblasts and PBMC cells, heat shock proteins, proteins affected by interferons, cytoskeletal proteins, and the presence of antibody against protein in human sera. Additional information has been entered for the cell cycle-regulated and DNA replication protein cyclin (PCNA). Information entered in the PBMC database includes molecular weight and potential markers for sorted populations of lymphocyte subtypes. For those proteins that have been matched to AMA proteins, information contained in some entries may be transferred from the AMA database.

Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis.

We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific MoAbs, and separated to a high degree of homogeneity by FACS into CD4+ helper T cells, CD8+ suppressor T cells, CD20+ B cells, and N901 (NHK-1)+ NK cells. The four lymphocyte subpopulations were labeled with [35S]methionine for 14 hr, solubilized in lysis buffer, and analyzed by two-dimensional gel electrophoresis (IEF). Of about 1000 proteins resolved in each case, most were found to be common to all subpopulations. However, eight putative markers for B1+ (proteins 5525, Mr = 63,700; 5621, Mr = 63,700; 8311, Mr = 36,900; 2202, Mr = 36,300; 6121, Mr = 30,300; 106, Mr = 29,300; 5009, Mr = 23,000; 8012, Mr = 11,600) and one for N901+ (protein 8129, Mr = 30,400) were identified. In contrast, no major protein markers were found that could differentiate T4+ and T8+ cells from each other or from B cells and NK cells. With the exception of two B1+ markers (proteins 5525 and 5621), lower but variable levels of the other markers were observed in all cell types. All the putative protein markers have been identified in the protein database of human peripheral blood mononuclear cells (PBMCs) (see accompanying article by Celis et al.). Comparison of the overall patterns of protein synthesis of the unsorted PBMCs with those of the four subpopulations showed that the synthesis of some major PBMC proteins decreased substantially in the sorted subsets. These proteins are most likely not of monocyte origin, as these cells constituted only about 15% of the total PBMCs. Also, the inhibition does not seem to be due to the addition of the single MoAbs or to cell cycle differences. Taken together, the data provide a background for further studies of protein profiles in normal (resting or activated) and malignant hematopoietic cells.