The PROCLIPI international registry of early-stage mycosis fungoides identifies substantial diagnostic delay in most patients.

BACKGROUND: Survival in mycosis fungoides (MF) is varied and may be poor. The PROCLIPI (PROspective Cutaneous Lymphoma International Prognostic Index) study is a web-based data collection system for early-stage MF with legal data-sharing agreements permitting international collaboration in a rare cancer with complex pathology. Clinicopathological data must be 100% complete and in-built intelligence in the database system ensures accurate staging. OBJECTIVES: To develop a prognostic index for MF. METHODS: Predefined datasets for clinical, haematological, radiological, immunohistochemical, genotypic, treatment and quality of life are collected at first diagnosis of MF and annually to test against survival. Biobanked tissue samples are recorded within a Federated Biobank for translational studies. RESULTS: In total, 430 patients were enrolled from 29 centres in 15 countries spanning five continents. Altogether, 348 were confirmed as having early-stage MF at central review. The majority had classical MF (81·6%) with a CD4 phenotype (88·2%). Folliculotropic MF was diagnosed in 17·8%. Most presented with stage I (IA: 49·4%; IB: 42·8%), but 7·8% presented with enlarged lymph nodes (stage IIA). A diagnostic delay between first symptom development and initial diagnosis was frequent [85·6%; median delay 36 months (interquartile range 12-90)]. This highlights the difficulties in accurate diagnosis, which includes lack of a singular diagnostic test for MF. CONCLUSIONS: This confirmed early-stage MF cohort is being followed-up to identify prognostic factors, which may allow better management and improve survival by identifying patients at risk of disease progression. This study design is a useful model for collaboration in other rare diseases, especially where pathological diagnosis can be complex.

Folliculotropic mycosis fungoides: clinicopathological analysis of 17 patients.

BACKGROUND: Folliculotropic mycosis fungoides (FMF) represents a variant of MF characterized by hair follicle invasion of mature, CD4-positive small lymphoid cells with cerebriform nuclei. The disease displays resistance to standard treatment modalities and has an unfavourable course. OBJECTIVE: Clinical analysis of 17 patients with FMF collected between 2005 and 2012, investigation of tumour cells and involved hair follicle. METHODS: Re-evaluation of clinical data, wide panel immunohistochemistry investigation on paraffin-embedded biopsy material, T-cell receptor gene rearrangement analysis of the samples. RESULTS: Male and older age group predominance, frequent head-neck involvement, acneiform lesions, keratotic plugs, cysts, nodules, follicular papules, alopecia and classic mycosis fungoides-like plaques represented the main clinical characteristics. Treatment response showed a wide range from transient complete response to therapy resistance and death due to the disease. The pathological alterations: folliculotropism, mild epidermotropism, follicular plugging, mucinous degeneration of hair follicle, basaloid hyperplasia, syringotropism were similar to those observed previously. The first case of a CD8-positive folliculotropic mycosis fungoides - with unusual clinical presentation - is reported here. Nestin overexpression of mesenchymal cells of the isthmic and suprabulbar regions of hair follicle and the reappearance of dermal nestin-expressing cells were observed in association with immature dendritic cell hyperplasia. Altered CK19 expression was detected suggesting a potential role of follicular keratinocytes in the disease process. It was found that a proportion of neoplastic T cells constantly express programmed death-1 receptor in our patients contrary to classic mycosis fungoides. CONCLUSION: The spectrum of the clinical manifestation and the course of folliculotropic mycosis fungoides are broad and differ from classic mycosis fungoides. Folliculotropic neoplastic T-cell proliferation is associated with activation of inflammatory reactive T- and B-lymphoid cells, mesenchymal cells and changes in the hair follicle.

Salicylic acid treatment via the rooting medium interferes with stomatal response, CO2 fixation rate and carbohydrate metabolism in tomato, and decreases harmful effects of subsequent salt stress.

Salicylic acid (SA) applied at 10(-3) m in hydroponic culture decreased stomatal conductance (g(s)), maximal CO(2) fixation rate (A(max) ) and initial slopes of the CO(2) (A/C(i)) and light response (A/PPFD) curves, carboxylation efficiency of Rubisco (CE) and photosynthetic quantum efficiency (Q), resulting in the death of tomato plants. However, plants could acclimate to lower concentrations of SA (10(-7) -10(-4) m) and, after 3 weeks, returned to control levels of g(s), photosynthetic performance and soluble sugar content. In response to high salinity (100 mm NaCl), the pre-treated plants exhibited higher A(max) as a function of internal CO(2) concentration (C(i) ) or photosynthetic photon flux density (PPFD), and higher CE and Q values than salt-treated controls, suggesting more effective photosynthesis after SA treatment. Growth in 10(-7) or 10(-4) m SA-containing solution led to accumulation of soluble sugars in both leaf and root tissues, which remained higher in both plant parts during salt stress at 10(-4) m SA. The activity of hexokinase (HXK) with glucose, but not fructose, as substrate was reduced by SA treatment in leaf and root samples, leading to accumulation of glucose and fructose in leaf tissues. HXK activity decreased further under high salinity in both plant organs. The accumulation of soluble sugars and sucrose in roots of plants growing in the presence of 10(-4) m SA contributed to osmotic adjustment and improved tolerance to subsequent salt stress. Apart from its putative role in delaying senescence, decreased HXK activity may divert hexoses from catabolic reactions to osmotic adaptation.

Richter's and prolymphocytic transformation of chronic lymphocytic leukemia are associated with high mRNA expression of activation-induced cytidine deaminase and aberrant somatic hypermutation.

Chronic lymphocytic leukemia (CLL) is an indolent B-cell non-Hodgkin's lymphoma that may transform into higher-grade lymphoma. The transformation involves an increased number of prolymphocytic cells, termed prolymphocytic transformation (PLT) or the development of diffuse large B-cell lymphoma (DLBL), also referred to as Richter's transformation (RT). To analyze whether activation-induced cytidine deaminase (AID), which is essential for somatic hypermutation (SHM) of normal B-cells, and malfunction of SHM termed aberrant somatic hypermutation (ASHM) are associated with higher-grade transformation of CLL, AID mRNA expression and the mutation pattern of c-MYC, PAX-5 and RhoH genes were analyzed in eight cases of CLL without transformation and in 21 cases that showed RT or PLT. Chronic lymphocytic leukemia cases, which showed no transformation or eventually transformed into higher-grade lymphoma, showed low levels of AID mRNA expression and low frequency of mutations of c-MYC, PAX-5 and RhoH genes. In both RT and PLT, high-levels of AID mRNA expression and high-frequency mutations of c-MYC, PAX-5 and RhoH genes were detected. These results indicate that AID expression and ASHM are associated with higher-grade transformation of CLL and provide further evidences that AID expression and ASHM may be activated during the clonal history of B-cell lymphomas.

Clonal selection in the bone marrow involvement of follicular lymphoma.

To characterize the pathways of bone marrow (BM) involvement of follicular lymphoma (FL), we performed morphological and immunophenotypical analysis of tumor cells from lymph nodes (LNs) and corresponding BMs in 21 patients with FL. In three cases, genealogical trees were constructed based on the immunoglobulin variable region heavy chain (IgV(H)) gene sequences of tumor clones from LNs and BMs. Results showed that FLs within the BMs display identical or lower cytological grades than in the LNs. In the majority of cases, different proportions of tumor cells expressed bcl-2, CD10 and Ki67 in LNs and BMs. Tumor cells in the BM showed ongoing somatic hypermutation of the IgV(H) genes; the distribution of these mutations was highly consistent with antigen selection. The topology of the genealogical trees revealed that different subclones populate the LN and BM and BM infiltration may occur at different points of the clonal evolution of FL. Early descendants of the original tumor clone and derivatives of diversified tumor clones may invade the BM. These results suggest that the BM involvement of FL is associated with intensive clonal selection of tumor cells, and the BM provides a microenvironment similar to the germinal centers of LNs, where tumor cells retain their biological nature.

Relationship between the mutational status of VH genes and pathogenesis of diffuse large B-cell lymphoma in Richter's syndrome.

Patients with chronic lymphocytic leukemia (CLL) may develop diffuse large B-cell lymphoma (DLBL), also known as Richter's syndrome. Mutational status of immunoglobulin (Ig) heavy-chain variable region (VH) genes have prognostic impact in CLL. Patients with mutated VH genes have a stable disease, whereas patients with unmutated VH gene have more aggressive disease. The mutational status of CLLs that transform to DLBL is unknown. To reveal whether Richter's syndrome occurs in CLLs with mutated or unmutated VH genes, we have performed mutational analysis on serial specimens from eight patients. CLL and DLBL tumorclones were identical in five cases and they were different in three cases. Six CLLs expressed unmutated and two cases expressed mutated VH genes. In five of the six unmutated CLLs, the DLBL clones evolved from CLL tumorclones and the VH genes expressed by DLBLs were also unmutated. In one unmutated and two mutated CLLs, the DLBLs expressed mutated VH genes, but in these three cases the DLBL tumorclones developed as independent secondary neoplasm. These results suggest that Richter's syndrome may develop in both mutated or unmutated CLLs, but clonal transformation of CLL to DLBL occur only in the unmutated subgroup of CLL.

Microsatellite instability and hMLH1 promoter hypermethylation in Richter's transformation of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an indolent B cell non-Hodgkin lymphoma (NHL) that may transform into diffuse large B cell lymphoma (DLBL). This transformation is referred to as Richter's syndrome or transformation. To analyze whether microsatellite instability (MSI) and DNA mismatch repair defects are associated with Richter's transformation, we have performed microsatellite analysis, mutational analysis of hMLH1 and hMSH2 genes and methylation status analysis of CpG island of the hMLH1 promoter on serial biopsy specimens from 19 patients with CLL. Ten cases of CLL showed no histologic alteration in the second biopsy, and nine cases of CLL underwent morphologic transformation to DLBL in the second biopsy. Using eight microsatellite loci, high level of MSI was associated with Richter's transformation in four cases of CLL, but none of the CLLs displayed this level of MSI without transformation. Mutations of the hMLH1 or hMSH2 genes were not detected in any of the lymphoma samples. In five cases of Richter's transformation the hMLH1 promoter was hypermethylated in both CLL and DLBL samples. Hypermethylation of the hMLH1 promoter associated with high-level of MSI in four cases, and low-level of MSI in one case. These results suggest that in certain cases of Richter's transformation the DNA mismatch-repair defect-initiated genetic instability may play a role in tumor progression.

Hepatocyte proliferation and cell cycle phase fractions in chronic viral hepatitis C by image analysis method.

OBJECTIVE: Chronic hepatitis is characterized by necrosis of liver cells, accompanied by an inflammatory reaction and compensatory cell proliferation. The interaction of the core and non-structural proteins of hepatitis C virus (HCV) with several cellular factors suggests that cell proliferation may be influenced by HCV. The aim of this study was to investigate hepatocyte proliferation and DNA ploidy patterns in patients with chronic viral hepatitis C (CH-C) compared with chronic non-viral hepatitis (CH-N), using a TV image analysis method. METHODS: The DNA index (DI) and cell phase fractions (G1, S, G2) were measured by means of digital picture analysis method on nuclear suspensions of Feulgen stained hepatocytes. Cells were taken from the liver biopsy specimens of 71 patients with CH-C and 24 patients with CH-N. Twenty-six normal liver samples were used as controls. RESULTS: Significantly higher G1 (94 +/- 4) and lower S (3.56 +/- 3.16) phase fractions were measured in CH-C compared with CH-N (G1, 90 +/- 6; S, 6.4 +/- 5.99). The DI of moderate (1.12 +/- 0.05) and severe (1.12 +/- 0.05) CH-C showed near-aneuploid DNA content, while diploidy (DI < 1.10) was detected in cases of CH-N. CONCLUSION: The higher G1 and lower S cell cycle phase fractions in CH-C reflect decreased hepatocyte proliferation compared with CH-N. The near-aneuploid DNA content of the HCV-infected liver samples may be a sign of increased genetic instability, which may contribute to the carcinogenic potential of HCV.

Effect of hepatitis C virus on hepatocyte proliferation and DNA ploidy in patients with chronic hepatitis C.

Hepatitis C virus infection may act as a cofactor by inducing chronic hepatitis and cirrhosis, playing a promoting role in the multistep process of hepatocarcinogenesis by maintaining liver inflammation, hepatocyte necrosis and regeneration. The aim of this study was to measure the DNA ploidy and cell proliferation of hepatocytes in patients with chronic hepatitis C. Hepatocyte nucleus suspension was analyzed from 45 patients with chronic hepatitis C and from 27 patients with chronic hepatitis non-C. The histopathological pattern of chronic hepatitis samples/grade, stage/was investigated. A significantly lower cyclin A protein expression and cytometrically measured S-phase fraction was observed in chronic hepatitis C as compared to chronic hepatitis non-C, representing suppressed cell proliferation of virus infected cells. In the chronic hepatitis C groups, the S-phase fraction depression was moderate, the grade of inflammation and cyclin A protein expression were also decreased, mainly in the severe grade group. In chronic hepatitis non-C, the number of cyclin A staining-positive cells increased parallel with severity of the inflammation. In addition, the HCV infection caused a near diploid minimally aneuploid cellular DNA content in the cases of moderate and severe histological groups. In contrast, the cellular DNA content was consequently diploid-independent of histological grades in chronic hepatitis non-C. Our results suggest that in chronic viral hepatitis C, the hepatocyte proliferation is suppressed parallel with the degree of inflammation, while the DNA content becomes aneuploid. The aneuploidy is a sign of genetic instability, predisposing the affected cells to unbalanced chromosomal abnormality which finally leads to malignant transformation.

Expression of transforming growth factor-alpha in hepatoblastoma.

BACKGROUND: Transforming growth factor-alpha (TGF-alpha) is a potent stimulator of cell proliferation in the liver and in liver tumors; however, its significance and association with hepatocyte proliferation remains unclear. METHODS: Expression of TGF-alpha and proliferation markers, such as proliferating cell nuclear antigen (PCNA) and cyclin A, were studied and correlated with each other in samples of tumor and surrounding liver tissue taken from nine patients with hepatoblastoma. An avidin-biotin-peroxidase immunohistochemical method was used for detection of TGF-alpha, PCNA, and cyclin A, and in situ hybridization was used to detect TGF-alpha mRNA. RESULTS: Two types of tumor cells of epithelial origin were distinguished based on the expression of TGF-alpha protein and RNA. The more differentiated "fetal" phenotype had a high expression of TGF-alpha and correlated with a low expression of proliferation markers. The less differentiated "embryonal" phenotype had low TGF-alpha expression and high proliferation activity. CONCLUSIONS: The expression of TGF-alpha is associated with a certain morphologic phenotype of tumor cells in hepatoblastoma; higher expression can be detected in more differentiated tumor cells. The negative correlation between the expression of TGF-alpha and proliferation markers suggests that the less differentiated embryonal cells do not depend on growth stimulation provided by TGF-alpha.

[Amantadine in the prevention of nosocomial influenza]. / A nosocomialis influenza amantadin prophylaxisa.

Amantadine prophylaxis was performed in 91 patients during influenza A epidemics. It was used for patients with chronic heart, pulmonary and metabolic disease, for immundeficienty and elderly patients. Patients with gravidity, lactation, epilepsy, peptid ulcer or serious liver disorder were excluded from prophylaxis. The daily dose was 200 mg, which was reduced to 100 mg in people over 65. The chemoprophylaxis was combined with killed influenza vaccine in 6 patients. No influenza-like illness occurred among patients with prevention. Light side-effect was observed in 5 patients. Ten peoples who were excluded from prophylaxis caught serologic proven influenza. Amantadine prophylaxis is appropriate for prevention of nosocomial influenza among high-risk patients in institutions because of other diseases.

Differential regulation of the synthesis and activity of the major cyclin-dependent kinases, p34cdc2, p33cdk2, and p34cdk4, during cell cycle entry and progression in normal human T lymphocytes.

Three major cyclin-dependent kinases, p34cdc2, p33cdk2, and p34cdk4 were examined in normal human T cells stimulated to enter the cell cycle in vitro. None of the three genes was expressed in resting T cells. Transcripts form the cdk4 and cdk2 genes were detectable as early as 3 and 8 hr after stimulation, respectively, whereas cdc2 gene transcripts were not detectable until about 24 hr, shortly before S phase entry. Immunoblot analysis showed that resting T cells contained little p34cdk4, no p34cdc2, and a low level of p33cdk2 protein. Increased amounts of p34cdk4, p33cdk2, and p34cdc2 proteins were seen at about 7, 10, and 30 hr after stimulation, respectively. Immunoprecipitates of each of the kinases were assessed for histone H1 kinase activity. Activity due to p33cdk2 first became detectable in mid-G1 phase and increased dramatically after entry into S phase. Active p34cdc2 kinase was not detected until about 40 hr after stimulation, about 10 hr after the first appearance of the protein. Immunoprecipitates of p34cdk4 possessed almost no H1 histone kinase activity; however, activity was detected as early as 10 hr after cell activation when a protein (p60Rb) derived from the retinoblastoma susceptibility gene product was used as substrate. Cells were synchronized about the G1/S and G2/M borders by aphidicolin and nocodazole. Cells arrested prior to S-phase contained high levels of active p33cdk2 and essentially no active p34cdc2, despite the fact that large amounts of both proteins were present. Cells arrested by nocodazole had high levels of active p34cdc2 and greatly reduced levels of p33cdk2 kinase activity. The results suggest that the major role for the p34cdc2 kinase is at mitosis, whereas that for p33cdk2 is in late G1 and/or S phase. The p34cdk4 protein, present in aphidicolin-blocked cells, was nearly absent from cells arrested at the G2/M border; however, kinase activity was low in cells blocked at both points, suggesting that the major role for p34cdk4 may be in G1 phase.

Symmetry of the activation of cyclin-dependent kinases in mitogen and growth factor-stimulated T lymphocytes.

The entry of resting T cells into the G1 phase of the cell cycle after stimulation by mitogens is controlled by a series of biochemical events that are independent of growth factors. These events follow the initial signals stimulated through the engagement of the T-cell receptor and include activation of the cyclin-dependent kinases Cdk6, Cdk4, and Cdk2, as well as a transient phosphorylation of the retinoblastoma gene product (p110Rb) by one or several of these proteins. A progression signal such as that delivered by interleukin-2 then induces a second phase of Cdk6, Cdk4, and Cdk2 activation, along with sustained phosphorylation of p110Rb in the activated T cells. This second signal is required to carry the cells into the S phase and beyond. Quantitative and qualitative differences in the expression and activity of these proteins may be critical to maintain the delicate balance that is necessary to ensure the normal progression of T cells through the cell cycle.

Effects of iron-depletion on cell cycle progression in normal human T lymphocytes: selective inhibition of the appearance of the cyclin A-associated component of the p33cdk2 kinase.

Iron removal by the chelating-agent deferoxamine (DFO) arrests cell cycle progression of activated human T cells in late G1 phase, before the G1/S border. The effects of the drug on molecules that regulate progression through the cell cycle were defined. DFO (10 mumol/L) inhibited induction of transcription of the cdc2 gene, but had no effect on accumulation of cdk2, cdk4, or interleukin (IL)-2-transcripts. No detectable p34cdc2 protein accumulated, but synthesis of the p33cdk2 protein was begun. It accumulated to normal levels during the first 20 to 30 hours of incubation in the presence of DFO. Furthermore, p33cdk2 was activated as an H1 histone kinase. As active p33cdk2 primarily represents complexes of the p33 protein with cyclin E or cyclin A, the effects of DFO on these cyclins were examined. Although the induction of synthesis and early accumulation of cyclin E and cyclin E-associated kinase activity appeared normal, the appearance of cyclin A and cyclin A-associated kinase activity were inhibited by DFO. However, the production of cyclin A mRNA appeared to be normal in the presence of DFO. A major effect of DFO in blocking cell cycle progression may be mediated through inhibition of the appearance of cyclin A protein and, therefore, a major component of p33cdk2 activity. The results also indicate that the p33cdk2/cyclin E activity produced in the presence of DFO was not sufficient for completion of the G1 phase of the cell cycle.

Regulation of synthesis and activity of the PLSTIRE protein (cyclin-dependent kinase 6 (cdk6)), a major cyclin D-associated cdk4 homologue in normal human T lymphocytes.

The PLSTIRE protein (cyclin-dependent kinase 6 (cdk6)), which shares extensive sequence homology (approximately 70%) with cdk4, was identified as the earliest inducible member of the cdk family of proteins in human T lymphocytes induced to proliferate in vitro by stimulation either with phorbol 12,13-dibutyrate and ionomycin (PDB/I) or PHA. The p40cdk6 protein was present in resting cells and increased amounts were detected 6 h after stimulation. It increased in amount throughout the first cell cycle but was present in reduced amounts at later times. Activity of the kinase, determined by in vitro phosphorylation of recombinant truncated retinoblastoma tumor suppressor gene (Rb) protein (p60Rb), paralleled p40cdk6 protein amounts. Cyclins D2 and D3 were the major cyclins associated with p40cdk6, with D2 predominating in early G1 phase. Both PDB and ionomycin were required for maximal accumulation of p40cdk6, but either agent alone stimulated some increase in amount and activity of the protein. p40cdk6 also increased in amount in cells activated in the presence of cyclosporin A or FK506, drugs that inhibit production of IL-2 and cell proliferation, suggesting that initial induction occurred independently of IL-2-mediated cell cycle progression. Furthermore, increased accumulation of p40cdk6 protein and activity occurred in cells rendered "competent" (responsive to IL-2) by a brief treatment with PDB/I. Thus, increased accumulation of the protein and its activity begin before IL-2/IL-2 receptor interaction, suggesting that the cdk6-cyclin D2 complex might be involved in acquisition of the competent state in human T lymphocytes.

Differential requirements for interleukin-2 distinguish the expression and activity of the cyclin-dependent kinases Cdk4 and Cdk2 in human T cells.

We examined the expression and activity of Cdk4 and Cdk2 in resting, competent, and proliferating normal human T cells. Expression of Cdk4 but not of Cdk2 was induced in competent T cells independent of an IL-2 signal. This up-regulation of Cdk4 mRNA and protein was resistant to the immunosuppressant drugs cyclosporin A (CsA) and FK506. A further increase in Cdk4 expression was seen upon stimulation of competent T cells by IL-2, as was de novo expression of Cdk2. Cyclin D2, a Cdk4 partner, showed similar patterns of regulation as Cdk4. The increases in Cdk4 and cyclin D2 expression seen in competent T cells were functionally significant since Cdk4 immunoprecipitates from these cells phosphorylated recombinant RB protein in vitro. Despite the lack of an increase in the expression of Cdk2, a small pool of pre-existing Cdk2 protein detected in resting T cells could be activated upon induction of competence. These data demonstrate that 1) the signals that lead to induction of competence in T cells stimulate an IL-2-independent and CsA-resistant phase of Cdk4 and cyclin D2 expression, Cdk4 kinase activity, and Cdk2 kinase activity, and 2) IL-2 stimulates a second phase of Cdk4 and cyclin D2 expression and de novo expression of Cdk2 in these cells. The data show that the expression and activity of these major cell cycle regulatory proteins are controlled differentially by growth factors and indicate a role for Cdk4 and cyclin D2 in T-cell cycle entry and/or early G1 progression and for Cdk2 in later G1 progression and G1/S transition.

Association of proliferating cell nuclear antigen with cyclin-dependent kinases and cyclins in normal and transformed human T lymphocytes.

The proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and appears to be needed for both DNA synthesis and DNA repair. It is present in low amount in resting normal human T lymphocytes and, upon mitogenic stimulation with phorbol dibutyrate and ionomycin, begins to increase in mid-G1 phase, approximately 12 to 15 hours before entry into S phase. PCNA continues to increase in amount throughout the cell cycle and remains high in proliferating cultures. PCNA was extracted from activated normal T cells and from the transformed T-lymphoblastoid cell line Jurkat by a method that recovered approximately 98% of total cellular PCNA but yet retained its associations with other proteins. PCNA immunoprecipitates possessed H1 histone kinase activity, which increased in parallel with increasing cellular content of PCNA. Both the cdc2 and cdk2 kinases were found associated with PCNA in normal T cells, in amounts consistent with detected kinase activity. The results indicate that PCNA is not an inhibitory molecule of cdk/cyclin activity. Both normal and transformed T cells contained PCNA in association with cdk2, cdk4, cdk5, and cdk6, with the amount of PCNA associated with these molecules increasing in the order listed. Relatively high amounts of PCNA were also found associated with cyclins D2 and D3, the major cyclin partners of cdk6 in T cells. Though detected in normal cells, PCNA/cdc2 complexes were present in exceedingly low amount, if at all, in Jurkat cells. This cell line appeared to contain more of nearly all of the cdk and cyclin molecules analyzed, but there seemed to be little difference in the patterns of association of these molecules with PCNA in the cell line as compared with normal human T cells.

Release from G0/G1 arrest induced by dimethyl sulfoxide in human lymphoid cells: regulation of synthesis and activation of the p33cdk2 and p34cdc2 kinases.

Raji cells, a human Burkitt's lymphoma-derived cell line, will accumulate in a G0-like state upon prolonged (5-6 days) incubation in medium containing 1.5% dimethyl sulfoxide (DMSO). After removal of DMSO, the cells reenter the cell cycle in a synchronous manner and proliferate. After 5.5 days incubation in DMSO, S phase entry occurs at about 21-24 h after release, which is about the length of the first G1 phase of normal human lymphocytes which are stimulated in vitro to enter the cell cycle. The G0-like state of arrested cells and the sequence of events occurring after release from DMSO mimic, in most ways studied, those of normal lymphocytes. Arrested Raji cells lack many cell cycle-regulated molecules, including cyclin A, proliferating cell nuclear antigen, and the p34cdc2 kinase. They contain only hypophosphorylated p110Rb and a low level of enzymatically inactive p33cdk2 kinase. After reentering the cell cycle, a series of events occurred, including phosphorylation of p110Rb and accumulation of the cyclin A and proliferating cell nuclear antigen proteins in mid-G1 and the accumulation of the p33cdk2 and p34cdc2 proteins beginning in late G1, just prior to S-phase entry. Cyclin E levels in Raji cells appeared to be less regulated than in normal cells, with high levels of this protein being present in resting cells and throughout the entire cell cycle. The time courses of activation of the p34cdc2 and p33cdk2 kinases were similar; both became detectable at about 21 h after release and increased greatly in early S.(ABSTRACT TRUNCATED AT 250 WORDS)

Role for Ca2+ in expression of cell cycle regulated genes in PAF-stimulated cells.

Platelet-activating factor (PAF) is a powerful stimulator of a wide variety of cells. In transformed human B-lymphoblastoid cell lines, PAF increases intracellular Ca2+ concentrations ([Ca2+]i) and induces the expression of the proto-oncogenes c-fos and early growth response gene-2 (EGR2). Here, we present data that evaluates the role of Ca2+ in the PAF-dependent induction of these cell-cycle activated genes. PAF (10(-7) M) increased c-fos and EGR2 mRNA levels in cells suspended in Ca(2+)-containing medium by 6-10-fold. In PAF-stimulated cells suspended in medium depleted of Ca2+, eliminating Ca2+ influx but not intracellular store release of Ca2+, the induction of gene expression was reduced by approx. 50%. In contrast, buffering of Ca2+ released from intracellular stores but maintaining transmembrane Ca2+ uptake had little effect on gene expression. When both sources of Ca2+ were eliminated, PAF-stimulated expression of these genes was completely prevented. This was not due to any toxicity to the cells since the response to phorbol ester under identical conditions was unaffected. The regulation of c-fos mRNA expression was paralleled by changes in levels of FOS protein. These data indicate that changes in [Ca2+]i, primarily from stimulated entry across the plasma membrane and to a lesser extent release of Ca2+ from sequestered intracellular stores, play an essential role in PAF-dependent triggering of c-fos and EGR2 mRNA expression.