[Effect of ectodysplasin-A1 on proliferation and cell cycle of ameloblast-like cell].

Objective: To investigate the effects of ectodysplasin-A1 (EDA1) on the proliferation and cell cycle of ameloblast-like epithelial cells (LS8 cells). Methods: Wild EDA1 plasmid pCR3-Flag-EDA1-W (wild group), syndrome mutant EDA1 plasmid pCR3-Flag-EDA1-H252L (mutant group) and empty vector plasmid pCR3-Flag (control group) were transfected into LS8 cells. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay and cell cycle was detected by flow cytometry. All tests were repeated three times. Results: Compared with the control group (0.105±0.032), the proliferation activity of the wild group (0.201±0.009) was significantly higher after 72 h (P<0.05). Compared with the control group (0.168±0.054) and the mutant group (0.194±0.059), the proliferation activity of the wild group (0.386±0.066) was significantly higher after 96 h (P<0.05). There was no significant difference between the mutant group and the control group at all time points (P>0.05). In the G0/G1 phase, compared with the control group (65.4%±2.1%) and the mutant group (66.6%±3.1%), the cell distribution ratio of the wild group (51.2%±1.1%) was significantly lower (P<0.01). In the S phase, compared with the control group (23.1%±2.0%) and the mutant group (21.9%±1.8%), the cell distribution ratio of the wild type group (37.3%±2.4%) was significantly higher (P<0.01). There was no significant difference in cell cycle distribution between the mutant group and the control group (P<0.05). Conclusions: Wild EDA1 promotes the proliferation of LS8 cells and the transformation from G0/G1 to S phase. The syndrome mutant EDA1 (EDA1-H252L) loses its function of regulating the cell proliferation and cell cycle of LS8 cells.

Codon 201(Gly) polymorphic type of the DCC gene is related to disseminated neuroblastoma.

The deleted in colorectal carcinoma (DCC) gene is a potential tumor-suppressor gene on chromosome 18q21.3. The relatively high frequency of loss of heterozygosity (LOH) and loss of expression of this gene in neuroblastoma, especially in the advanced stages, imply the possibility of involvement of the DCC gene in progression of neuroblastoma. However, only few typical mutations have been identified in this gene, indicating that other possible mechanisms for the inactivation of this gene may exist. A polymorphic change (Arg to Gly) at DCC codon 201 is related to advanced colorectal carcinoma and increases in the tumors with absent DCC protein expression. In order to understand whether this change is associated with the development or progression of neuroblastoma, we investigated codon 201 polymorphism of the DCC gene in 102 primary neuroblastomas by polymerase chain reaction single-strand conformation polymorphism. We found no missense or nonsense mutations, but a polymorphic change from CGA (Arg) to GGA (Gly) at codon 201 resulting in three types of polymorphism: codon 201(Gly) type, codon 201(Arg/Gly) type, and codon 201(Arg) type. The codon 201(Gly) type occurred more frequently in disseminated (stages IV and IVs) neuroblastomas (72%) than in localized (stages I, II, and III) tumors (48%) (P=.035), and normal controls (38%) (P=.024). In addition, the codon 201(Gly) type was significantly more common in tumors found clinically (65%) than in those found by mass screening (35%) (P=.002). The results suggested that the codon 201(Gly) type of the DCC gene might be associated with a higher risk of disseminating neuroblastoma.

The expression and antigenicity identification of recombinant rat TGF-beta1 in bacteria.

In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.

Mechanisms of differential activation of target gene promoters by p53 hinge domain mutants with impaired apoptotic function.

Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest functions that have been shown to be separable activities of p53. We report here that some mutants in the p53 hinge domain, a short linker between the DNA binding and tetramerization domains, differentially activated the promoters of p53 target genes and possessed an impaired apoptotic function. Our results indicate that the hinge domain may play an important role in differentially regulating p53 cell cycle arrest and apoptotic functions. However, the mechanisms by which p53 hinge domain mutants differentially activate its target genes, e.g. p21(WAF1/CIP1) and Bax, remain unknown. To investigate the possible mechanisms, recombinant p21(WAF1/CIP1) and Bax promoters were constructed, resulting in rearrangement of the existing p53 binding sites within a given promoter or actually swapping p53 binding sites between the two promoters. Our results suggest that multiple mechanisms of differential transactivation occur, depending on the molecular nature of the relevant hinge domain mutant, such as the possibility that dual separate DNA binding sites in the p21(WAF1/CIP1) promoter are responsible for the selective transactivation activity of p53 hinge domain mutant del300-327, which has a large deletion in the hinge domain. Lack of ideal p53 binding sites in the Bax promoter results in less potent activation than that seen with the p21(WAF1/CIP1) promoter when it is transactivated by hinge domain point mutant mutR306P or short deletion mutant del300-308 proteins. How the single mutation or the short deletion affect the conformation of p53 and consequently the transactivation of the Bax promoter will require further investigation of the relevant p53 protein: DNA-binding domain by NMR and x-ray crystallographic techniques.

Evaluation of germ-cell kinetics in infertile patients with proliferating cell nuclear antigen proliferating index.

AIM: To explore the usefulness of proliferating cell nuclear antigen proliferating index (PCNA PI) in the pathological diagnosis and treatment of male infertility. METHODS: Testicular biopsy specimen obtained from 48 cases of male infertility and 2 normal controls were fixed and embedded. The sections were stained with anti-PCNA monoclonal antibodies or haematoxylin/eosin. Proliferating index (PI), expressed as the percentage of germ-cell nuclei positively stained with PCNA antibody, was assessed from more than 20 seminiferous tubules or 600 germ-cells. RESULTS: The infertile patients were divided into 4 groups: Group 1, normal spermatogenesis (14 cases); Group 2, hypospermatogenesis (16 cases); Group 3, germinal arrest (10 cases); Group 4, Sertoli cell only syndrome (8 cases). The PCNA PI of normal control testis was 86.5% (mean value). Group 3 had a significantly lower PCNA PI (29.8%) than normal testis; Group 1 and 2 had similar PIs (82.3% and 82.3%, respectively) as the control testis. PI of the negative control (Group 4) was 0 as no germ-cells were found. CONCLUSION: PCNA PI is useful for assessing germ-cell kinetics, especially for pathological diagnosis of germinal arrest which is difficult to differentiate by routine HE staining technique. In germinal arrest, there is a significantly lowered PCNA PI, which is an indication of DNA synthesis deterioration, suggesting the use of therapies be different from those for hypospermatogenesis.

Reduced or absent expression and codon 201Gly/Arg polymorphism of DCC gene in rhabdomyosarcoma and Ewing's sarcoma/PNET family.

Genetic alterations occurring in various chromosomes have been described in many human tumors. The DCC gene was originally identified in colorectal cancer and was reported as a tumor suppressor gene that might be related to tumor metastasis. We investigated 10 cell lines and 15 fresh tumors of childhood rhabdomyosarcoma, 7 cell lines of Ewing's sarcoma, and 4 cell lines of primitive neuroectodermal tumor (PNET) for the expression and mutation of DCC gene by RT-PCR analysis and PCR-single stranded conformation polymorphism (SSCP) analysis. Twenty-five pairs of primers were used for PCR-SSCP. Six of ten (60%) cell lines of rhabdomyosarcoma and 3 of 7 (43%) cell lines of Ewing's sarcoma showed reduced or absent expression of DCC gene. There was no mobility shift within 24 exons by SSCP analysis, although 3 types of polymorphism were found at codon 201 in exon 3. Direct sequencing of different bands showed types I, II, and I/II representative of codon 201Gly, codon 201Arg, and codon 201Gly/Arg, respectively. The proportion of type I between fresh rhabdomyosarcoma and normal controls was not significant. Our results suggested that the inactivation of DCC gene may play a role in the pathogenesis of a subset of rhabdomyosarcoma and Ewing's sarcoma.

p53 mutants have selective dominant-negative effects on apoptosis but not growth arrest in human cancer cell lines.

A bidirectional expression vector that allowed equal transcription of cloned wild-type and mutant p53 cDNAs from the same vector was developed. The vector was transfected into CaLu 6 lung carcinoma cells or Saos-2 osteosarcoma cells. All p53 mutants examined were recessive to wild-type p53 transactivation of p21(WAF1/CIP1) but dominant-negative for transactivation of Bax. An examination of effects on growth arrest and apoptotic pathways indicated that all mutants were recessive to wild type for growth arrest but only three of seven mutants were dominant negative for induction of apoptosis.

Lack of homozygously inactivated p73 in single-copy MYCN primary neuroblastomas and neuroblastoma cell lines.

We examined 18 neuroblastoma cell lines and 32 primary single-copy MYCN tumor specimens to determine whether mutations of p73, a novel p53-related gene located in chromosome band 1p36.33, contribute to the genesis or progression of childhood neuroblastoma. By fluorescence in situ hybridization, 16 of the 18 cell lines, but only 3 of the 32 primary tumors, had evidence of a deleted p73 allele. Sequence analysis of the p73 coding region in the mRNAs expressed by these cell lines and tumors did not reveal inactivating mutations, suggesting that p73 is not homozygously inactivated in neuroblastoma. However, several novel splice forms of p73 mRNAs were identified, including one without exon 11 that predominated in multiple MYCN-amplified cell lines. Its encoded p73 protein differed from other splice forms in that the C-terminus was derived from an alternative reading frame. Further study of the functional properties of the protein encoded by this splice form of p73 will be needed to determine whether it contributes to the pathogenesis of childhood neuroblastoma with MYCN gene amplification.

The effect of Epstein-Barr virus gene BHRF1 expression on radioresistance of nasopharyngeal carcinoma cells.

In order to investigate the effect of the expression of Epstein-Barr virus gene BHRF1 on the apoptotic resistance of nasopharyngeal carcinoma cells to radiation, a highly expressing vector for BHRF1 was constructed and transfected into the nasopharyngeal carcinoma cell line CNE2. Then, the biologic alterations of the cells were tested after 60Co radiation. The results showed that, in the BHRF1-expressing cells, the apoptotic index was far lower than in the control groups after 60Co radiation, and cells recovered faster from the radiation, with a higher cell-proliferative rate, stronger ability of colony formation and tumor development in nude mice than that in the control groups. Given the functional homology of BHRF1 with bcl-2, our data indicate that BHRF1 expression could prohibit nasopharyngeal carcinoma cellular apoptosis caused by radiation and in this way contribute to oncogenic transformation.

Expression of osteopontin mRNA in normal and stone-forming rat kidney.

Human urine contains several macromolecules which inhibit calcium oxalate crystallization. Osteopontin (or uropontin), a secreted phosphoglycoprotein with the amino acid sequence Arg-Gly-Asp (RGD) and high affinity to hydroxyapatite, is one such inhibitor. To investigate the action of this protein on renal stone formation, the expression osteopontin gene in normal and chemically induced urolithiasis rat kidney was compared at both mRNA and protein levels. Northern blot analysis shown a significant increase of osteopontin mRNA level in stone-forming rat kidney compared with normal ones. In an in situ hybridization study, we localized the transcripts of the osteopontin gene in epithelial cells of both distal and collective tubules, and found a remarkably strong signal in stone-forming rats. The amount and distribution of the protein in kidney from immunocytochemistry staining showed the same pattern as seen in situ hybridization. These findings indicate that osteopontin may be an important macromolecule in the normal endogenous defence against the formation of urinary calculi.

Expression and mutational analysis of the DCC, DPC4, and MADR2/JV18-1 genes in neuroblastoma.

Loss of heterozygosity (LOH) on chromosome 18q21 is found frequently in various human cancers. Three candidate tumor suppressor genes, DCC (deleted in colorectal carcinomas), DPC4 (deleted in pancreatic carcinomas, locus 4), and MADR2/JV18-1 (MAD-related gene 2), have been cloned and identified from this chromosome region. We have reported recently that LOH on chromosome 18q is observed frequently in neuroblastoma. Alterations of DCC are involved in many human tumors. DPC4 and MADR2/JV18-1 are recently demonstrated to be altered in pancreatic and colorectal cancers, respectively. To confirm if inactivation of the DCC, DPC4, and MADR2/JV18-1 genes is involved in the pathogenesis of neuroblastoma and to clarify the mechanism of inactivation, we analyzed the expression of DCC, DPC4, and MADR2/JV18-1 in neuroblastoma cell lines and primary tumors by reverse transcription-PCR and investigated the mutations in the coding regions of these genes by PCR/reverse transcription-PCR single-strand conformation polymorphism. We found that 12 of 25 (48%) cell lines and 14 of 32 (44%) primary tumors, including 3 with 18q LOH, had absent or reduced expression of DCC mRNA. Expression was more likely to be reduced in advanced (67%) than in early stage neuroblastomas (24%) (P = 0.036), suggesting that inactivation of the DCC gene plays an important role in the progression of neuroblastoma. Altered expression of DPC4 was found in six (24%) cell lines and six (19%) tumors. MADR2/JV18-1 expression was reduced or absent only in four (16%) cell lines and three (9%) tumors. Mutations of the DCC genes were examined in 25 of 29 exons in neuroblastoma cell lines, and those exons in which mutations were found were further examined in primary tumors. We found missense mutations of AAC (Asn) to AGC (Ser) at DCC codon 176 in one cell line and ACC (Thr) to ATC (Ile) at codon 1105 in one cell line and tumor, respectively; polymorphisms of CGA (Arg) to GGA (Gly) at codon 201 and TTT (Phe) to TTG (Leu) at codon 951 in most of the cell lines and tumors; and a silent mutation of GAG (Glu) to GAA (Glu) at codon 118 in four cell lines and five primary tumors. We did not identify any mutations in the DPC4 and MADR2/JV18-1 genes in neuroblastoma. Our results suggested that mutations of the DCC gene may be involved in the pathogenesis of neuroblastomas but failed to account for the relatively high frequency of the altered expression, implying that other mechanisms are responsible for the inactivation of the DCC gene in neuroblastoma. Low frequency of reduced or absent mRNA expression and lack of mutations in DPC4 and MADR2/JV18-1 genes suggested a limited role for these two genes in neuroblastoma.

Consistent detection of TLS/FUS-ERG chimeric transcripts in acute myeloid leukemia with t(16;21)(p11;q22) and identification of a novel transcript.

16;21 translocation is a recurrent primary abnormality in acute myeloid leukemia (AML). The genes involved in this translocation are ERG on chromosome 21 and TLS/FUS on chromosome 16. The rearrangement of the two chromosomes forms the TLS/FUS-ERG fusion gene and produces a consistent chimeric transcript on the der (21) chromosome. In this study, we analyzed the clinical characteristics of 19 patients with t(16;21)-AML, including 2 patients who evolved from myelodysplastic syndrome, and detected the chimeric transcripts of the TLS/FUS-ERG fusion gene in the patients during various clinical stages by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. We found that the patients with t(16;21) are characterized by a relatively younger age (median age, 22 years old), involvement of various subtypes of French-American-British classification and a poor prognosis: 18 of the 19 patients died of the disease (median survival was 16 months). Four types of TLS/FUS-ERG chimeric transcripts including a novel type were noted in the RT-PCR analysis. The novel transcript contained an additional 138 nucleotides consisting of TLS/FUS exon 8 and ERG exons 7 and 8 and had an in-frame fusion. These chimeric transcripts were consistently detectable in the samples obtained not only at diagnosis and relapse but also in short and long complete remission, suggesting that t(16;21)-AML is resistant to conventional chemotherapy. Thus, we recommend that t(16;21) should be monitored by RT-PCR even in clinical remission and the patients should be treated by other more powerful modality like stem-cell transplantation in the first remission.

Construction of retroviral vectors to induce a strong expression of human class interferon gene in human hepatocellular carcinoma cells in vitro.

AIM: To establish the hepatoma cell-specific expression of human interferon (IFN) gene mediated by retroviral vectors METHODS: Human interferon α and interferon ß complementary DNA (IFN cDNA) were cloned into the polylinker site of pMNSM retroviral vector to construct recombinant retroviral vectors pMNSIFNA and pMNSIFNB, with the transcription of IFN gene being driven by Simian virus 40 early region promoter (SV40) early region promoter. IFN cDNAs were also cloned into pMNAIFNA, pAMNSIFNA, and pMNAIFNB, with the transcription of IFN gene being driven by SV40 early region promoter regulated by α-fetoprotein enhancer. Next, the retroviral constructs were introduced into retroviral amphotropic packaging cells using the lipofectamine-mediated gene transfer procedure. The rate of plasmid transfection was (4-40) × 10(3) colonies/µg DNA/10(6) PA317 cells. The rate of retrovirus infection was (5-500) × 10(4) colony forming units (CFU)/mL. Further, the recombinant retroviruses were used to infect human hepatoma cells, renal carcinoma cells, and melanoma cell lines in the presence of 4 µmg/L polybrene. RESULTS: Northern and Dot hybridization of total RNA from the neomycin-resistant colonies and IFN expression assay indicated that human α fetoprotein enhancer induced efficient and specific transcription and expression of IFN genes driven by the promoter of different origins in human hepatoma cells, leading to high production of α fetoprotein. CONCLUSION: Cis active element of α-fetoprotein gene can drive specific expression of IFN genes in human hepatoma cells, which provides some valuable data for the hepatoma-specific immune gene therapy.

Alterations of the tumour suppressor gene DCC in neuroblastoma.

The deleted in colorectal carcinoma (DCC) gene, a candidate tumour suppressor, might be inactivated in a number of human cancers. In order to evaluate the possible role of DCC alterations in the pathogenesis of neuroblastoma, we examined 25 neuroblastoma cell lines and 16 primary tumours, including 6 samples with loss of heterozygosity (LOH) at the DCC locus for DCC mRNA expression, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. The level of DCC expression was significantly reduced or undetectable in 12 of 25 (48%) cell lines and 7 of 16 (44%) primary tumours, suggesting that inactivation of the DCC gene is involved in the development of neuroblastoma. Three of the 6 tumours with LOH at the DCC locus revealed reduced DCC mRNA expression, indicating that LOH at the DCC locus might have affected the levels of DCC mRNA. We also screened for mutations in 4 exons of the DCC gene in 12 cell lines by using PCR-single strand conformation polymorphism (PCR-SSCP) analysis. Point mutations were not found except a polymorphic change at codon 201. The mechanism for inactivation of the DCC gene will be further investigated.

Epstein-Barr virus BHRF1 prohibits the cells of nasopharyngeal carcinoma from apoptosis.

Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). The BHRF1 EBV protein is expressed at high levels in productively infected cells and certain latently infected cells. In order to investigate the effect of expression of BHRF1 on the biological behaviour of NPC cells, we constructed the BHRF1 high expression vector and transfected it into the NPC cell line, CNE2. Then, the alteration of proliferation and apoptotic rates in the cells were tested before and after camptothecin treatment. After treatment by camptothecin, BHRF1-CNE2 cells could constantly and slowly proliferate and its apoptotic rate was less than in control groups, and the number of cells in the G phase decreased and in the S phase increased. So, it suggests that BHRF1 expression can enhance the resistibility of CNE2 cells to DNA-damaging agents that cause apoptosis.

Study of the prevalence of diabetes mellitus in adults in the Shougang Corporation in Beijing.

The aim was to determine the burden of diabetes mellitus (DM) in an urban area of China to aid us in planning preventive measures for those at risk of DM. A survey was conducted among the 29,859 subjects aged between 30 and 64 belonging to 32 units of the Shougang Corporation (a heavy industry enterprise) within the Beijing area. WHO study protocols and diagnostic criteria were used to determine the prevalence of DM and impaired glucose tolerance (IGT). The results showed that the age-adjusted prevalence of DM and impaired glucose tolerance (IGT) was 3.63% and 4.19%, respectively, both increasing with age. Peak prevalence for both occurred in the 60-64 age group. Prevalence showed no difference between the sexes in DM but was higher for females in IGT. Obesity, being overweight, a family history of diabetes mellitus and in women, a history of delivering babies with macrosomia, all correlated closely with the prevalence of DM and IGT. High protein intake was also associated with DM, Smoking had no effect on either DM or IGT. Intellectual workers had a higher incidence of IGT than manual workers. Seventy per cent DM was undiagnosed prior to the survey. This survey, done according to the recommendation of WHO, and including appropriate adjustments, reflects the growing prevalence of DM and IGT in this population. It can be compared with other studies for epidemiological analysis.

Preliminary study of the frequency of benign prostatic hyperplasia and prostatic cancer in China.

OBJECTIVES: This study determined the frequency of benign prostatic hyperplasia (BPH) and cancer of the prostate (CaP) in China. METHODS: Prostate specimens from 321 unselected autopsies were collected from 1989 to 1992. Slices were cut vertically every 0.5 cm from apex to base. Five to 12 slices were obtained from each prostate. Specimens were stained with hematoxylin-eosin and Masson trichrome. Sixty surgical specimens obtained from cystoprostatectomies with intact prostate were included to determine the frequency of latent CaP. RESULTS: The frequency of BPH, by age, was as follows: 41 to 50 years, 13.2%; 51 to 60 years, 20%; 61 to 70 years, 50%; 71 to 80 years, 57.1%; 81 to 90 years, 83.3%. The frequency of latent CaP, by age, was as follows: 41 to 50 years, 2.2%; 51 to 60 years, 9.3%; 61 to 70 years, 5.9%; 70 years or older, 25%. Incidental CaP was found in 4.9% (33 of 676) of BPH surgical specimens. The incidence of and mortality from CaP in Beijing were 2.41 per 100,000 men and 1.19 per 100,000 men, respectively, between 1985 and 1987. CONCLUSIONS: BPH was rare in China in the early years of this century, but it has become a common disease in recent decades. The histologic frequency of BPH in China was similar to that in Western countries, but the histologic frequency of latent CaP was less than half that in Western countries. The incidence of and mortality from CaP in China are about 20 times less than those in Western countries. Histologic CaP in a Chinese man is not as likely to evolve into clinical CaP as in a Western man.

[Modulation effect of cimetidine on the production of IL-2 and interferon-gamma in patients with gastric cancer].

The modulation effects of cimetidine on the production of IL-2 and IFN-gamma by the peripheral blood mononuclear cells in 31 patients with gastric cancer and 32 normal subjects were studied. Their T lymphocyte subsets were also assayed. The IL-2 and IFN-gamma activity in patients were significantly lower than that in normal subjects (P < 0.01). Cimetidine could significantly promote IL-2 and IFN-gamma production. There was a significant negative correlation between OKT8 subsets and the activity of IL-2 and IFN-gamma (P < 0.01). The results showed that cimetidine could be used as an adjunct in the treatment of advanced neoplasm.

[The extracellular matrix and liver disease: a study with enzymoimmunological assay].

Serum concentrations of laminin and hyaluronate were assayed in 138 patients of liver disease with an enzymoimmunological method. There was a mild increase of hyaluronate level in patients with acute hepatitis (P < 0.05) and a significant increase of both serum laminin and hyaluronate concentrations in patients with chronic hepatic diseases, as compared with those in healthy controls (P < 0.001). Serum laminin and hyaluronate reached the highest levels in patients with liver cirrhosis. Comparing the results from a group of patients with liver cirrhosis with those from a reference group of patients with chronic active hepatitis, we obtained values for specificity, sensitivity, and diagnostic efficiency of 0.90, 0.90 and 0.90 respectively. The results suggested that quantification of serum laminin and hyaluronate may be a useful test to assess the degree of chronic liver injury and to diagnose liver fibrosis.