High CC chemokine receptor 7 expression improves postoperative prognosis of lung adenocarcinoma patients.

BACKGROUND: Chemokines and chemokine receptors not only have significant roles in cancer metastasis and tumorigenesis but also act as antitumour agents. The interaction between the Crk-like adaptor protein (CrkL), which is encoded by the CRKL gene, and non-receptor tyrosine kinase c-ABL is reported to transform many cells into malignant cells. We examined the effects of CC chemokine receptor 7 (CCR7), CCR7 ligands and CrkL and c-ABL in lung adenocarcinoma. METHODS: One hundred and twenty patients with lung adenocarcinoma were included in this historical cohort analysis. We examined CCR7 and CCR7 ligands and CrkL and c-ABL mRNA expressions in surgically resected lung adenocarcinoma specimens and evaluated their contribution to prognosis, and the relationship with epidermal growth factor receptor (EGFR) and TP53 mutations. RESULTS: High CCR7 mRNA expressions indicated better prognoses than those of the groups with low CCR7 mRNA expressions (P=0.007, HR=2.00, 95% CI of ratio: 1.22 -3.31). In lung adenocarcinoma, CrkL and c-ABL mRNAs were related to CCR7 mRNA expression (P<0.0001). CrkL and c-ABL mRNA expressions were influenced by EGFR mutations. A high expression of CCL19 was a good prognostic factor of lung adenocarcinoma. CONCLUSION: We propose that CCR7 and CCL19 are clinically good prognostic factors and that CCR7 is strongly related to CrkL and c-ABL kinase mRNA expression in lung adenocarcinoma.

Unexpected serum level of vancomycin after oral administration in a patient with severe colitis and renal insufficiency.

OBJECTIVE: To report a case in which the serum concentration of vancomycin (VCM) reached the supratherapeutic range following oral administration in a patient with severe pseudomembranous colitis and renal insufficiency. CASE SUMMARY: A 65-year-old, 70 kg weighing man with severe acute pancreatitis and acute renal failure was subjected to continuous hemodiafiltration (CHDF). CHDF could only be performed intermittently because of the unstable circulation dynamic of this patient. After admission, intravenous VCM therapy was initiated. Thereafter, oral VCM administration was begun (0.5 g every 6 h). Despite the discontinuation of intravenous VCM after the first 2 days of oral VCM, the serum VCM concentration increased gradually to 49.8 mg/l over a period of 2 weeks from the initiation of oral administration (34.4 mg/l). Based on pharmacokinetic analysis, the bioavailability of VCM was estimated to over 33%. Autopsy findings indicated broadly distributed necrosis on the lamina propria of the mucosa throughout all parts of the intestine below the duodenum. DISCUSSION: This case indicates necessity of the careful monitoring after oral high-dose VCM administration in a patient with a broadly distributed necrosis and renal insufficiency. CONCLUSIONS: TDM should be considered according to renal function, the severity of enteritis and the total dosage of oral VCM administration.

Enhancement of chemosensitivity toward peplomycin by calpastatin-stabilized NF-kappaB p65 in esophageal carcinoma cells: possible involvement of Fas/Fas-L synergism.

Chemosensitivity to anticancer drugs was compared between two human esophageal carcinoma cell lines, T.Tn and YES-6 cells. T.Tn cells were more resistant than YES-6 cells to peplomycin (PEP) but not to the other anticancer drugs such as camptothecin, mitomycin C and cytosine arabinoside. Western blot analysis showed higher expression levels of m-calpain and activated mu-calpain in T.Tn cells than in YES-6 cells. On the other hand, YES-6 cells showed a high expression level of calpastatin, which is a calpain-specific endogenous inhibitor. To investigate whether calpain activity was involved in the chemosensitivity, T.Tn cells were transfected with calpastatin cDNA in an inducible expression vector. The induction of calpastatin was accompanied by increased chemosensitivity to PEP. The increases in calpastatin levels were followed by serial increases in the expression levels of NF-kappaB p65 and Fas. Since purified m- or mu-calpain degraded NF-kappaB p65 in vitro, it is possible that calpastatin suppressed calpain-mediated degradation of NF-kappaB p65. Fas ligand (Fas-L) protein levels increased after treatment of the parental T.Tn and calpastatin-transfected cells with PEP, suggesting the synergism between calpastatin-induced Fas and PEP-induced Fas-L. These results suggest that calpain/calpastatin expression levels are effective markers for predicting the sensitivity of human esophageal carcinoma cells to PEP.

Immunoglobulin heavy chain variable region (VH) genes of B cell chronic lymphocytic leukemia cells from lymph nodes show somatic mutations and intraclonal diversity irrespective of follicular dendritic cell network.

We analyzed the immunoglobulin heavy chain variable region (VH) gene in 4 Japanese cases of B cell chronic lymphocytic leukemia (B-CLL) with enlarged lymph nodes to clarify the presence of somatic mutations and intraclonal diversity. We also attempted to determine the role of the follicular dendritic cell (FDC) network in some proliferation centers, where tumor cells are mitotically active. Immunohistochemical studies revealed that all 4 cases showed the typical immunophenotype: CD5+, CD23+, IgM+ and IgD+. DNA was extracted from paraffin sections (lymph node) and rearranged VH gene was amplified by PCR. All but one exhibited a moderate number of somatic mutations, with percentages ranging from 4.1 to 9.5, and one of which indicated the effect of antigen selection on its VH gene. Multiple clone analysis of whole tissues showed intraclonal diversity in one case, whose VH gene carried a somatic mutation but the effect of antigen selection was not apparent. We further examined microdissected tissues to elucidate the relationship between FDC network and VH gene status in 2 cases. In one case, intraclonal diversity was not apparent irrespective of FDC network, however, both tumor cells around the FDC network and those apart from the FDC showed signs of intraclonal diversity in another case, suggesting that intraclonal diversity was not related to the FDC network in B-CLL. Here we demonstrate that some cases of B-CLL involved in lymph node carried mutated VH genes and showed intraclonal diversity like the tumor cells in the peripheral blood. However, the significance of the FDC network in the proliferation center still remains to be resolved.

Hypermethylation of the CD44 gene is associated with progression and metastasis of human prostate cancer.

BACKGROUND: CD44 is a metastasis suppressor gene for prostate cancer and the down-regulation of CD44 and its variants is associated with the progression of prostate cancer. Also, hypermethylation of the CpG islands of the CD44 gene is closely associated with transcriptional inactivation, resulting in the decreased expression of CD44. To clarify the exact role of methylation status of CpG islands of CD44 gene in the progression and metastasis of prostate cancer, we investigated the methylation status of this gene in primary and metastatic human prostate tumors obtained from surgery or autopsy. METHODS: We examined 97 samples from 40 Japanese patients with adenocarcinoma of the prostate. Tumor tissues were obtained from radical prostatectomy specimens from eight patients with stage B, 12 patients with stage C and three patients with stage D1 and at autopsy from 17 hormone-refractory metastatic cases, who had initially responded to the therapy and thereafter relapsed. Distant metastatic tissues were also obtained at autopsy (i.e., liver, lung, kidney, mammary gland, and pelvic lymph nodes) from 10 of 17 hormone-refractory cases. We analyzed the hypermethylation status of CD44 promotor region by PCR using genomic DNAs digested with the m(5)C-sensitive restriction enzyme HpaII. RESULTS: The correlation between the methylation status of CD44 gene and the stage progression of prostate cancer was statistically significant (P = 0.0438). In two of 10 hormone-refractory cases, a comparison of the methylation status of the CD44 gene in metastases to that in primary tumors revealed interfocal heterogeneity of CD44 methylation status. CONCLUSIONS: These results indicate an important role of CD44 methylation in the progression and metastasis of prostate cancer, although the amount of methylational heterogeneity is substantial among metastatic sites within the same patient.

A human protein with sequence similarity to Drosophila mastermind coordinates the nuclear form of notch and a CSL protein to build a transcriptional activator complex on target promoters.

Mastermind (Mam) has been implicated as an important positive regulator of the Notch signaling pathway by genetic studies using Drosophila melanogaster. Here we describe a biochemical mechanism of action of Mam within the Notch signaling pathway. Expression of a human sequence related to Drosophila Mam (hMam-1) in mammalian cells augments induction of Hairy Enhancer of split (HES) promoters by Notch signaling. hMam-1 stabilizes and participates in the DNA binding complex of the intracellular domain of human Notch1 and a CSL protein. Truncated versions of hMam-1 that can maintain an association with the complex behave in a dominant negative fashion and depress transactivation. Furthermore, Drosophila Mam forms a similar complex with the intracellular domain of Drosophila Notch and Drosophila CSL protein during activation of Enhancer of split, the Drosophila counterpart of HES. These results indicate that Mam is an essential component of the transcriptional apparatus of Notch signaling.

Activation of mitogen-activated protein kinase through alpha5/beta1 integrin is required for cell cycle progression of B progenitor cell line, Reh, on human marrow stromal cells.

OBJECTIVE: Attachment to bone marrow (BM) stromal cells is crucial for the normal growth and development of B-cell progenitors (pro-B). However, the molecular mechanisms by which contact facilitates the proliferation of pro-B cells are not completely understood. This study was performed to investigate this interaction. MATERIALS AND METHODS: A model pro-B cell line (Reh) and a human BM stromal cell line (KM102) were used. Flow cytomery was used for cell cycle analysis. Western Blotting and immunoprecipitation were utilized to examine the levels of cyclin-dependent kinase (cdk) and p27(Kip1). RESULTS: Attachment to both KM102 and normal BM stromal cells significantly promoted the growth of Reh cells. Pretreatment of Reh cells with anti-integrin beta1 or alpha5 monoclonal antibody (mAb), but not alpha4 or ICAM-1 mAb, abrogated this enhancement of proliferation. Furthermore, stroma attachment resulted in shortening of the G(1) phase of cell cycle, significant increases cdk2 activity, degradation of cdk inhibitor p27-GST protein, and decrease in levels of p27(Kip1) protein. In addition, solid-phase cross-linking of alpha5 via immobilized antibody also resulted in extracellular signal-regulated (ERK)-2 kinase phosphorylation, increase in cdk2 activity, decrease in levels of p27(Kip1) protein, and enhanced proliferation that was inhibited by treatment with PD98059, a specific ERK inhibitor. CONCLUSION: Integrin alpha5beta1-mediated stroma contact promotes the proliferation of B-cell progenitors through the activation of ERK-2, which in turn modulates cell cycle regulation machinery including induction of cdk2 activity and degradation of p27(Kip1) and contributing to acceleration of the G(1) phase of cell cycle progression.

Expression of cell cycle regulating proteins in an unusual transformation of mantle cell lymphoma.

We describe here two patients with mantle cell lymphoma (MCL) who after a few years, developed to the diffuse large cell lymphoma (DLCL) (anaplastic centrocytic lymphoma) growing in a diffuse sheets without the classical MCL component. In both the initial and second biopsy specimens, in each case, tumor cells were positive for cyclin D1, sIgM, sIgD, and CD5, but were negative for CD10 and CD23. In a study of immunoglobulin heavy chain (IgH) gene rearrangement, using the polymerase chain reaction (PCR) method, the products obtained from each paired biopsy tissue sample were the same size, and in one case had an identical sequence to the non-mutated VH gene. Immunohistochemistry was used to examine the expression of p53, p27Kip1 and cyclin E. Interestingly, there was clear overexpression of p53 protein in case 1 but not in case 2, compared with other typical MCL cases. The expression of p27Kip1 in the second biopsies of each case was decreased compared with those in the initial biopsies. In case 2, however, p27Kip1 was clearly expressed in the first and second biopsies, in contrast to other typical MCL cases. Thus these 2 cases demonstrate not only that the variant form of MCL may arise de novo, but also that MCL may transform to DLCL at the time of relapse. Although the mechanism of tumor progression/transformation is still poorly understood, the overexpression of p53 or p27Kip1 may be linked to a cellular mechanism involved in the development of the variant form of MCL.

Diffuse large B cell lymphoma expressing the natural killer cell marker CD56.

The expression of the natural killer (NK) cell antigen, CD56, in hematological malignancies is rare. However, there are several reports that some hematological malignancies, such as T/NK cell lymphoma, multiple myeloma (MM) and acute myeloid leukemia (AML), express this molecule. In B cell non-Hodgkin's lymphomas (NHL), however, very limited number of cases have been reported to express CD56 molecule. Although one study has recently described that half of microvillous B cell lymphoma (MVL), an uncommon subset of large cell lymphoma, expressed CD56, there have been no reports about most common type of B-NHL, diffuse large B cell lymphoma (DLBL) other than a mention of weak CD56 expression in one of 83 DLBL. We herein presented the first case of diffuse large B cell lymphoma expressing CD56 clearly. The immunophenotype determined by immunostaining and flow cytometric analysis was CD10+, CD19+, CD20+, CD45RO-, CD3- and CD56+. On immunohistochemical study, neither bcl-2 nor TIA-1 was positive for tumor cell. Monoclonal immunoglobulin heavy chain (IgH) gene rearrangement was detected, and the sequence analysis of the variable region of IgH (VH) suggested that this tumor was derived from antigen selected post germinal center B cell. Conventional combination chemotherapy (CHOP) was administered, and the patient has still been in complete remission for 10 months.

Clinicopathological evaluation of the Revised European-American Classification of Lymphoid Neoplasms (REAL) in Japan.

After the publication of a Revised European-American Classification of Lymphoid Neoplasms (REAL classification) in 1994, there have been reports from Europe and America regarding its practical utility and clinical significance. However, no studies have been published from Eastern countries including Japan. It has been well recognized that the distribution of malignant lymphoma in Japan is quite different from that seen in Western countries. In addition, some new entities have also been described in the REAL classification. Therefore, it seems important to examine its practical utility and clinical significance in Japan. Of the 579 cases reviewed, approximately 68% were B-cell non-Hodgkin's lymphoma (NHL) followed by 27% T-cell lymphomas. Hodgkin's disease (HD) comprised only 5% of all cases, making the ratio of NHL to HD 20.6. The most common type was diffuse large B-cell lymphoma which represented about 37% of all cases. Peripheral T-cell lymphomas, unspecified (PTCL), occurred in 15% whereas marginal zone B-cell lymphoma followed (14.9%). However, follicle center lymphoma (FCL) was less common (4.4%) as has been previously reported. We evaluated the clinical significance of the new REAL classification in 244 cases. International Prognostic Index (IPI) was a powerful predictor of survival (p<0.0001), and the immunophenotype was significant (p<0.05). Furthermore, here, we also attempt to establish a prognostic scheme based on the histologic type. In conclusion, the REAL classification appears to be useful and clinically significant in Japan.

Potential use of serum CD44 as an indicator of tumour progression in acute leukemia.

CD44 is a widely expressed cell surface glycoprotein with various functions. This molecule is shed from the cell surface and released as a soluble molecule. High serum levels of CD44 have been demonstrated in some solid tumours. In this study we measured serum CD44 in 25 patients with acute leukemia, in 12 with bacterial infections, and in 13 normal controls. The levels of serum CD44 of patients with bacterial infections were significantly higher (mean 531.3+/-60.1 ng/ml, p<0.001) than those of normal controls (299. 0+/-115.4 ng/ml). Acute leukemia patients before treatment had almost four-fold higher levels of serum CD44 than normal controls (mean 1301.9+/-1384.6 ng/ml, p<0.01). Serum CD44 levels were correlated with clinical status. After treatment the serum CD44 levels significantly decreased, but they were still higher than in normal controls. Patients in complete remission all relapsed if serum CD44 levels were higher than 500 ng/ml (normal+2 SD) after chemotherapy. The serum CD44 levels were correlated with the absolute numbers of leukemic cells in peripheral blood. The results demonstrated that serum CD44 levels correlate well with the clinical status of acute leukemia, and such evaluation may provide a reliable tumour marker of acute leukemia.

Expression of Cell Cycle Regulating Proteins in an Unusual Transformation of Mantle Cell Lymphoma.

We describe here two patients with mantle cell lymphoma (MCL) who after a few years, developed to the diffuse large cell lymphoma (DLCL)( anaplastic centrocytic lymphoma) growing in a diffuse sheets without the classical MCL component. In both the initial and second biopsy specimens, in each case, tumor cells were positive for cyclin D1, sIgM, sIgD, and CD5, but were negative for CD10 and CD23. In a study of immunoglobulin heavy chain (IgH) gene rearrangement, using the polymerase chain reaction (PCR) method, the products obtained from each paired biopsy tissue sample were the same size, and in one case had an identical sequence to the non-mutated VH gene. Immunohistochemistry was used to examine the expression of p53, p27(Kip1) and cyclin E. Interestingly, there was clear overexpression of p53 protein in case 1 but not in case 2, compared with other typical MCL cases. The expression of p27(Kip1) in the second biopsies of each case was decreased compared with those in the initial biopsies. In case 2, however, p27(Kip1) was clearly expressed in the first and second biopsies, in contrast to other typical MCL cases. Thus these 2 cases demonstrate not only that the variant form of MCL may arise de novo, but also that MCL may transform to DLCL at the time of relapse. Although the mechanism of tumor progression/transformation is still poorly understood, the overexpression of p53 or p27(Kip1) may be linked to a cellular mechanism involved in the development of the variant form of MCL.

Role of p27Kip1 and cyclin-dependent kinase 2 in the proliferation of non-small cell lung cancer.

The cell cycle is governed by a family of cyclin-dependent kinases (Cdks). Cdk2 forms a functional complex with cyclin E and plays a pivotal role in the regulation of G1/S transition. Cdk2 activity is negatively regulated by interactions with inhibitors. p27Kip1, one of the most potent inhibitors of Cdk2, was recently identified as a powerful negative prognostic marker in non-small cell lung cancer as well as in colorectal and breast cancer. In the present study, the expression of p27 and Ki-67 antigen in nonneoplastic and cancerous lung tissues was determined by immunohistochemistry. After establishing that the antibody-measured p27 labeling index was a good reflection of the level of p27 expression measured by Western blotting, we show that p27 labeling index is decreased in cancerous lung tissues, compared with nonneoplastic lung tissues, and exhibits a significant inverse relation to the proliferation marker Ki-67 antigen, detected with monoclonal antibody MIB-1. Consistent with these data, all cancerous lung tissues showed enhanced degradation activity of p27 compared with nonneoplastic lung tissues and, in addition, increased levels of the phosphorylated form of Cdk2, as determined with Western blot analysis. The H1 histone kinase activity associated with Cdk2 was also increased in non-small cell lung cancers. Statistical analysis showed that proliferative activity as measured by MIB-1 labeling index was highly correlated with Cdk2 activity (r = 0.767, P < 0.0015). These results suggest that p27 and Cdk2 may play an important role in the proliferation of non-small cell cancer.

Expression of exon v6-containing CD44 isoforms is related to poor prognosis of acute myelocytic leukemia.

CD44 is a cell surface glycoprotein with a number of isoforms generated by alternative splicing of ten 'variant' exons in humans. Variant exon 6-containing isoforms of CD44 (CD44v6) have been implicated in the metastatic potential of rat carcinoma cell lines. Human homologues of CD44v6 are expressed in several tumour types and are involved in their progression. In the present study, we examined the expression of CD44 mRNA in 20 acute myelocytic leukemias by semiquantitative RT-PCR analysis and assessed its prognostic value. In all leukemic cells the predominant isoform was the 'standard' form of CD44 (CD44H), and intense bands were found in eight cases. CD44v6 was expressed in 11 cases, although its levels and those of other variants containing exon v7 through to v10 were much lower than those of CD44H. Isoforms containing exon v4 or v5 could not be detected. The expression of CD44v6 correlated with the death rate from leukemia (p > 0.05), but was not related to other risk factors. On the other hand, the intense expression of CD44H did not correlate with the prognosis of leukemia. CD44v6 thus appears to be a marker for the poor prognosis of acute myelocytic leukemia.

Interleukin-11 Gene Expression in Human Lymphoid Malignancy.

Interleukin-11 (IL-11) has diverse biological effects in hematopoiesis has been shown to share important functions with IL-6. However, unlike IL-6, there has been little information about the expression of IL-11 in lymphoid malignancy. Using reverse transcriptase polymerase chain reaction, IL-11 transcript was found in a number of lymphoid cell lines. A high level of expression was found in follicular lymphoma cell line FL18, and this was also detectable by Northern blotting. When TPA/A23187 were added to the culture of bone marrow stromal cell line KM102, IL-11 transcripts were rapidly upregulated. In contrast, levels of IL-11 transcripts were not increased in FL18 even upon the stimulation. The addition of actinomycin D to the cultures showed that the half life of the transcripts was similar in both FL18 and KM102. This suggests that posttran scriptional processes might not be involved in the constitutive expression of FL18. The results of IL-11 bioassay and enzymed-linked immunosorbent assay showed that FL18 did not secrete biologically active IL-11 into the medium. IL-11 transcript was also found in lymphoma cells in patient with malignant lymphoma, but not in B and T lymphocytes from reactive hyperplasia. Our results indicate that IL-11 transcripts can sometimes be produced in the neoplastic transformation of lymphoid cells.

12-O-tetradecanoylphorbol-13-acetate (TPA) downregulates expression of CD30 in erythroleukemia cell line K562.

CD30, a member of the tumour necrosis factor/nerve growth factor receptor superfamily, has been thought to have pleiotropic functions on immune response. However, there has been only a little information about the mechanism of CD30 expression. In this study, modulation of the CD30 molecule was investigated by the treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). When cultures were supplemented with TPA, CD30 transcript was downregulated in a dose- and time-dependent manner in the erythroleukemia cell line K562. Half reduction of CD30 transcript, precursor protein and surface protein was at 3 h, 6 h, and 40 h, respectively, by Northern blot and Western blot analyses. This consecutive reduction of both the transcript and proteins suggests that TPA directly inhibits the transcriptional step of CD30, and subsequently CD30 molecules would decrease on the cell surface. To determine whether the protein kinase C (PKC) pathway is involved in this reduction, a PKC inhibitor, 10 microM H-7, was added to the K562 culture. The addition of H-7 recovered the inhibitory effect of TPA, indicating that PKC is involved in the transcription of CD30. When either 2 micrograms/ml actinomycin D or 20 micrograms/ml cycloheximide was added simultaneously with TPA to the culture, the repressive effect of TPA on CD30 was abolished. These results showed that the repression would also partly involve ongoing mRNA and protein synthesis under TPA treatment.

The role of mel-18, a mammalian Polycomb group gene, during IL-7-dependent proliferation of lymphocyte precursors.

mel-18 is a mammalian homolog of Drosophila melanogaster Polycomb group genes. Mice lacking the mel-18 gene show a posterior transformation of the axial skeleton, severe combined immunodeficiency, and a food-passing disturbance in the lower intestine due to hypertrophy of the smooth muscle layer. In this study, the severe combined immunodeficiency observed in mel-18 mutant mice is correlated with the impaired mitotic response of lymphocyte precursors upon interleukin-7 stimulation. Strikingly, the axial skeleton and lymphoid phenotypes are identical in both mel-18 and bmi-1 mutants, indicating that the Mel-18 and Bmi-1 gene products might act in the same genetic cascade. These results suggest that mammalian Polycomb group gene products are involved in cell cycle progression in the immune system.