Primary carcinoid of the testis associated with carcinoid syndrome.

Testicular carcinoid is a rare disease accounting for less than 1% of all testicular neoplasms. It rarely manifests symptoms of carcinoid syndrome. Recent reports have noted that only 1.1-3.1% of testicular carcinoid tumors are complicated by carcinoid syndrome. In general, large tumor size and the presence of carcinoid syndrome are features associated with a malignant course. In the present case, pathological findings revealed pure carcinoid of the testis without metastasis. Moreover, watery diarrhea due to carcinoid syndrome disappeared and the serum serotonin level normalized following orchiectomy. The patient was followed up for 12 months with whole body computed tomography scan and assessment of serotonin levels. To date, there is no evidence of tumor recurrence. These findings suggest that monitoring serum serotonin levels may be useful as a marker during follow up of this type of tumor.

Trans-dominant inhibition of poly(ADP-ribosyl)ation potentiates alkylation-induced shuttle-vector mutagenesis in Chinese hamster cells.

In most eukaryotic cells, the catalytic activation of poly(ADP-ribose) polymerase (PARP) represents one of the earliest cellular responses to the infliction of DNA damage. To study the biological function(s) of poly(ADP-ribosyl)ation, we have established stable transfectants (COM3 cells) of the SV40-transformed Chinese hamster cell line C060 which conditionally overexpress the PARP DNA-binding domain upon addition of dexamethasone. We could demonstrate that DNA-binding domain overexpression, which leads to trans-dominant inhibition of poly(ADP-ribosyl)ation, potentiates the cytotoxicity of alkylation treatment and of gamma-radiation. Likewise, carcinogen-induced gene amplification, viewed as a manifestation of genomic instability, was potentiated by the overexpression of the PARP DNA-binding domain. Recently, we studied the effect of trans-dominant PARP inhibition on mutagenesis by employing a shuttle-vector assay in which mutagen-exposed plasmid pYZ289 is electroporated into COM3 cells. We could show that dexamethasone-induced overexpression of the PARP DNA-binding domain in COM3 cells potentiates the mutagenicity of the alkylating agent N-methyl-N-nitrosourea, while no effect of dexamethasone treatment on mutation frequency was recorded in control cells lacking the PARP DNA-binding domain transgene. Taken together, our results further substantiate the role of poly(ADP-ribosyl)ation in the maintenance of genomic integrity and stability under conditions of genotoxic stress.

Detection of circulating tumor cells in patients with prostate cancer using prostate specific membrane-derived primers in the polymerase chain reaction.

BACKGROUND: Prostate specific membrane antigen (PSM) is a transmembrane glycoprotein that has been described as human prostate specific. It is possible that PSM could be used as a biomarker for staging prostate cancer. METHODS: Peripheral blood mononuclear cells from 2 groups of patients with prostate cancer were used for the detection of PSM messenger ribonucleic acid (mRNA) using the reverse transcriptase-polymerase chain reaction (RT PCR) method. Group 1 consisted of 29 untreated patients (13 stage B cancer, 5 stage C, and 11 stage D cancers). Group 2 consisted of 40 treated patients (23 responded well and 17 had recurrence after treatment). In addition, blood specimens from 30 patients with benign prostatic hyperplasia, 5 women, and 5 men undergoing cystoprostatectomy for invasive bladder cancer were used as controls. We then correlated patient and disease characteristics with PCR assay results. RESULTS: Samples of all 40 controls were negative for PSM-mRNA. Thirteen of 29 patients of group 1 (45%) were positive for PSM-mRNA. The PCR positive rate did not correlate with clinical stage, pathologic stage, tumor grade, or serum PSA levels. Nine of 40 patients (22%) in group 2 were positive for PSM-mRNA, and the majority (8 of 9) were derived from the group of patients with recurrent disease. CONCLUSIONS: A nested RT PCR assay for PSM mRNA can detect circulating tumor cells in the peripheral blood of patients with prostatic cancer. These results suggest that the detection of circulating tumor cells could be useful for monitoring disease progression of prostatic cancer.

Expression of heparan sulfate proteoglycan mRNA in rat kidneys during calcium oxalate nephrolithiasis.

This study used reverse transcription polymerase chain reaction (RT-PCR) to examine heparan sulfate proteoglycan (HS-PG) mRNA expression levels during stone formation in the rat kidney. Total RNA in kidneys was extracted and converted to cDNA. PCR products were resolved by electrophoresis on 1.5% agarose gel and visualized with ethidium bromide. Fragment intensity and area were measured using an image analyzer. Control cyclophilin and HS-PG mRNAs were expressed in all samples examined as 235 bp and 506 bp bands, respectively. Cyclophilin expression in the normal group was not significantly different from expression in the group that formed stones. However, the level of HS-PG mRNA expression apparently increased in calcium oxalate (CaOx) microlith. The findings suggest an association between CaOx nephrolithiasis and expression of HS-PG in the rat kidney.

Higher susceptibility of erythropoietin-producing renal cell carcinomas to lysis by lymphokine-activated killer cells.

Erythropoietin production by renal cell carcinoma (RCC) is reported to be a potential marker for interleukin-2/interferon-alpha-responding tumor. We have investigated whether erythropoietin of RCC cells is involved in the immune recognition by lymphokine-activated killer (LAK) cells. Cells from primary culture of RCC cells expressing erythropoietin-mRNA or producing erythropoietin were more susceptible to lysis by LAK cells than those not expressing or producing it, respectively. RCC cells transfected with erythropoietin-cDNA became more susceptible to lysis by LAK cells than their erythropoietin-negative parental cells. These results indicate higher susceptibility of erythropoietin-producing RCC cells to lysis by LAK cells, suggesting that erythropoietin of RCC cells is involved in the immune recognition by LAK cells.

Expression of the MAGE gene family in human lymphocytic leukemia.

The MAGE gene family, encoding tumor-rejection antigens recognized by cytotoxic T lymphocytes, is frequently expressed in human solid cancers. However, its expression in leukemia has not been well studied. We have investigated MAGE gene expression at the mRNA level in human leukemia. The MAGE gene family was expressed in 17 of 34 (50%) examples of T cell leukemia (12/21 patients' peripheral blood mononuclear cells and 5/13 cell lines), in 7 of 16 (44%) cases of B cell leukemia (1/8 and 6/8 respectively), but in none of 23 myelomonocytic leukemia cases (0/16 and 0/7), as evaluated by the primers common to the MAGE-1, -3, -4 (-4a and/or -4b), and -6 genes and the semi-quantificative reverse transcription/polymerase chain reaction method. None of a panel of normal lymphoid cells expressed the MAGE gene family. As revealed by the primers specific for each of the MAGE genes, the MAGE-1, -2, -3, -4 or -6 gene was expressed in 8, 8, 6, 2, or 6 respectively out of 23 types of leukemia cell lines. Expression of the MAGE-1 protein in both the cell lines and patients' cells was confirmed by immunoblot analysis with the polyclonal antibody to recombinant MAGE-1 protein. Cellular MAGE-4 protein in the cell lines was measured by an enzyme-linked immunosorbent assay with the polyclonal and monoclonal antibodies to recombinant MAGE-4b protein. In summary, the MAGE gene family was found to be expressed in the substantial proportion of T cell leukemias, but in no case of myelomonocytic leukemia. Antigens coded by the MAGE gene family could be important molecules for understanding specific immunity against lymphocytic leukemia.

trans-dominant inhibition of poly(ADP-ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N'-nitro-N-nitrosoguanidine but does not limit DNA replication of a polyomavirus replicon.

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD+ serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible, high-level expression of the DNA-binding domain. Induction of this domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.

A newly designed experimental system for exposure of mammalian cells to extremely low frequency magnetic fields.

To examine the biological effects of extremely low frequency magnetic field (ELFMF), we have designed and manufactured a new equipment for long-term and high-density exposure of cells to ELFMF. The ELFMF exposure system consists of a generator of magnets with a built-in CO2 incubator, an alternating current (AC) power supply, a gas compressor and a thermocontroller for the incubator, and a cooling unit for the magnets. The CO2 incubator made of acrylic resin is inserted into the inner-space of the silicon steel strip-cores. In this system, the temperature of the incubator is maintained at 37 +/- 0.5 degrees C. The maximum magnetic flux density on the exposure area of the incubator is 500 mT (T; tesla) at a current of 556 Arms (rms; root mean square) at 50 Hz. The long-term (up to 120 hr) exposure of 400 mT ELFMF did not affect the growth of both HL60RG and CCRF-CEM cells originated from human leukemia. The post-X-irradiation exposure of 400 mT ELFMF for 2 hr also did not affect the radiation sensitivity of GM0637 and TAT2SF cells originated from a normal human and an ataxia telangiectasia patient.

Increase in the capability of interleukin 2 gene-transduced renal cell carcinoma cells to induce cytotoxic lymphocytes.

The immunogenicity of human cancer cells transfected with interleukin 2 (IL-2) gene, a potent vaccine candidate, has not yet been fully investigated. Human renal cell carcinoma (RCC) cells transduced with human IL-2 gene (RCC-IL-2) were investigated in vitro for the capability to induce lymphokine-activated killer (LAK) or cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocyte (TIL). The RCC-IL-2 cells stimulated PBMC to demonstrate LAK activity, and also stimulated autologous TILs to proliferate and exhibit cytotoxicity relatively restricted to autologous tumor cells. In contrast, both parental RCC and RCC transduced with neomycin gene alone failed to induce these activities. These results indicate that RCC-IL-2 cells are more potent than the other RCC cells with regard to inducing cytotoxic lymphocytes against autologous tumor cells.

Analysis of mutations caused by DNA double-strand breaks produced by a restriction enzyme in shuttle vector plasmids propagated in ataxia telangiectasia cells.

Rejoining of DNA double-strand breaks (DSB) produced by a restriction endonuclease AvaI in the supF gene in a plasmid pZ189Ava, and mutations presumably due to the altered rejoinings were analyzed. After allowing the rejoining and replication of the plasmids in human cells originating from normal subjects and ataxia telangiectasia (AT) patients, the plasmids were retrieved and those containing mutated supF were screened in an indicator strain of Escherichia coli. The proportion of correctly rejoined plasmids was significantly lower in AT cells than in normal cells, suggesting that AT cells have lower fidelity in rejoining DSB. DNA sequencing of the mutated supF genes revealed that all mutations were deletions or insertions occurring exactly or closely at the rejoining site in both normal and AT cells. In AT cells, the majority of mutations were deletions, while deletions and insertions were evenly formed in normal cells. AT cells may be deficient in the mechanism to protect the broken ends of DNA strands from the exonucleolytic digestion.

Transition of phenotypic dimorphism with regard to spontaneous sister chromatid exchange in Epstein-Barr virus-transformed Bloom's syndrome lymphoblastoid cell lines.

We recently established four lymphoblastoid cell lines (LCLs) by infecting the peripheral blood of four Japanese patients suffering from Bloom's syndrome (BS) with Epstein-Barr virus (EBV). During the course of propagating these cell lines, two of them exhibited dimorphism regarding spontaneous sister chromatid exchange (SCE), i.e., a mixed population consisted of cells with extremely high SCE levels characteristic of BS and cells with low SCE levels indistinguishable from that of normal control cells. On the other hand, the other two cell lines maintained a monomorphic population with high SCE levels at least until 30 weeks after EBV infection. The proportion of the cells with high SCE levels in the cell lines with dual phenotype declined as the population doubling numbers (PDN) increased with time and they became ultimately undetectable. The proportion of cells with low SCE levels at the time of EBV infection was estimated in one of these LCLs as 0.075% by extrapolating the linear regression of the logit for the proportion plotted against PDN. In view of the well-known stability of the monomorphic phenotype in representative BS LCLs during extended cultivation, together with the present observations on the dual phenotype, we conclude that the frequent establishment of BS LCLs exclusively with low spontaneous SCE levels is attributable to the various proportions of low-SCE cells existing in vivo in the B-lymphocytes pool of BS individuals and to the selective pressure against the high-SCE cells in in vitro cultures.

UV-induced base substitution mutations in a shuttle vector plasmid propagated in group C xeroderma pigmentosum cells.

To assess the contribution to mutagenesis of human DNA repair defects, the UV-irradiated shuttle vector plasmid pZ189 was propagated in fibroblasts derived from a xeroderma pigmentosum (XP) patient in DNA repair complementation group C. In comparison to results with DNA repair-proficient human cells (WI-38 VA13), UV-irradiated pZ189 propagated in the XP-C (XP4PA(SV)) cells showed fewer surviving plasmids and a higher frequency of mutated plasmids. Base sequence analysis of 67 mutated plasmids recovered from the XP-C cells revealed similar classes of point mutations and mutation spectrum, and a higher frequency of G:C to A:T transitions along with a lower frequency of transversions among plasmids with single or tandem mutations compared to plasmids recovered from the normal line. Most single-base substitution mutations (83%) occurred at G:C base pairs in which the 5'-adjacent base of the cytosine was thymine or cytosine. These results indicate that the DNA repair defects in XP-C, in comparison to data previously reported for XP-A, XP-D and XP-F, result in different UV survival and mutation frequency but in similar types of base substitution mutations.

Analysis of point mutations in an ultraviolet-irradiated shuttle vector plasmid propagated in cells from Japanese xeroderma pigmentosum patients in complementation groups A and F.

To assess the contribution to mutagenesis by human DNA repair defects, a UV-treated shuttle vector plasmid, pZ189, was passed through fibroblasts derived from Japanese xeroderma pigmentosum (XP) patients in two different DNA repair complementation groups (A and F). Patients with XP have clinical and cellular UV hypersensitivity, increased frequency of skin cancer, and defects in DNA repair. The XP DNA repair defects represented by complementation groups A (XP-A) and F (XP-F) are more common in Japan than in Europe or the United States. In comparison to results with DNA repair-proficient human cells (W138-VA13), UV-treated pZ189 passed through the XP-A [XP2OS(SV)] or XP-F [XP2YO(SV)] cells showed fewer surviving plasmids (XP-A less than XP-F) and a higher frequency of mutated plasmids (XP-A greater than XP-F). Base sequence analysis of more than 200 mutated plasmids showed the major type of base substitution mutation to be the G:C----A:T transition with all three cell lines. The XP-A and XP-F cells revealed a higher frequency of G:C----A:T transitions and a lower frequency of transversions among plasmids with single or tandem mutations and a lower frequency of plasmids with multiple point mutations compared to the normal line. The spectrum of mutations in pZ189 with the XP-A cells was similar to that with the XP-F cells. Seventy-six to 91% of the single base substitution mutations occurred at G:C base pairs in which the 5'-neighboring base of the cytosine was thymine or cytosine. These studies indicate that the DNA repair defects in Japanese XP patients in complementation groups A and F result in different frequencies of plasmid survival and mutagenesis but in similar types of mutagenic abnormalities despite marked differences in clinical features. These results, together with comparable studies from United States patients in XP complementation groups A and D, suggest that G:C----A:T somatic mutations may be important in the generation of human skin cancer by UV radiation.

Physical dosimetry at Nagasaki--Europium-152 of stone embankment and electron spin resonance of teeth from atomic bomb survivors.

Gamma-rays from thermal neutron-induced radionuclide of 152Eu in rocks near the ground center of the atomic bomb (A-bomb) explosion (hypocenter) in Nagasaki were measured with a pure germanium semiconductor detector. Depth profiles of 152Eu activity were obtained for 22 core samples taken from stone embankments on both sides of two rivers (the River Shimono-kawa and the River Urakami-gawa) within 500 m of the hypocenter since the activity in the rocks has a history of incident neutron energy. although the activities of the surface sections were varied from sample to sample, the slopes of depth profile in rock and the value of 152Eu activity in the depth of 240-280 mm were similar among the samples taken from the same location. On the other hand, neutron penetrating experiments using both a fast and a thermal neutron reactor were performed to obtain the relationship between the incident neutron energy spectra and the depth profiles of 152Eu activity in rock. The depth profiles in the bomb exposed samples were close to that obtained by using a 10 mm polyethelene moderator in the reactor experiments. Electron spin resonance (ESR) measurements from teeth of A-bomb survivors were carried out to estimate the individual gamma-ray dose of the survivors. The absorbed dose of ten tooth samples was estimated by ESR dosimetry. The results of ESR dosimetry were consistent with the calculations of tissue dose in air of A-bombs and irradiated shielding configuration of individuals.

Japanese patients with leukaemia following the use of Thorotrast including a patient with marked chromosomal rearrangements.

It is estimated that 20,000-33,000 persons have been injected with Thorotrast in Japan. By August 1984, 12 patients with leukaemia following the use of Thorotrast have been reported. Their clinical and haematological data indicate that most of the patients were males over 50 years of age and that the most frequent type of leukaemia involved the stem cells common to the granulocytic, erythroid and/or megakaryocytic lines. This assumption was supported by the evidence of marked chromosomal rearrangements in 100% of the cells from the bone marrow in our case. The median latent period from the administration of Thorotrast to the onset of leukaemia in Japan was 35 years, ranging from 16 to 45 years, which indicates that patients who were injected with Thorotrast should be carefully followed up in the future.