Antiproliferative effects of isoflavones on human cancer cell lines established from the gastrointestinal tract.

Seven isoflavones, biochanin A, daidzein, genistein, genistin, prunectin, puerarin, and pseudobaptigenin were tested for cytostatic and cytotoxic effects on 10 newly established cancer cell lines of the human gastrointestinal origin. Proliferation of HSC-41E6, HSC-45M2, and SH101-P4 stomach cancer cell lines was strongly inhibited by biochanin A and genistein, whereas other stomach, esophageal, and colon cancer lines were moderately suppressed by both compounds. Biochanin A and genistein were cytostatic at low concentrations (< 20 micrograms/ml for biochanin A, < 10 micrograms/ml for genistein) and were cytotoxic at higher concentrations (> 40 micrograms/ml for biochanin A, > 20 micrograms/ml for genistein). DNA fragmentation was observed at cytotoxic doses of both compounds, indicating the apoptotic mode of cell death by the compounds. Chromatin condensation and nuclear fragmentation of each cell line were also observed. The advent of apoptosis was dose dependent for both isoflavones. Biochanin A suppressed tumor growth of HSC-45M2 and HSC-41E6 lines in athymic nude mice. Our results suggest that two of isoflavone derivatives, biochanin A and genistein, inhibit the cell growth of stomach cancer cell lines in vitro through activation of a signal transduction pathway for apoptosis. Moreover, in vivo experiments demonstrate that biochanin A can be used as an anticancer agent.

Association of minisatellite instability with c-myc amplification and K-ras mutation in methylcholanthrene-induced mouse sarcomas.

Instability of microsatellite sequences are frequently found in human tumors. In addition, minisatellite sequences, another group of highly unstable sequences, serve as sensitive markers of genetic instability. We have studied minisatellite instability in methylcholanthrene-induced mouse sarcomas. These sarcomas frequently carry the amplified c-myc gene. Seven sarcomas without the amplification and seven others with the amplification were selected randomly. Regardless of the state of the c-myc gene amplification, these sarcomas exhibited a varying degree of transplantability in syngeneic mice. The hypervariable mouse minisatellite locus Ms6hm was found to be highly unstable, specifically among sarcomas with the amplified c-myc gene. However, chromosome instability, as analyzed by micronucleus assay, was observed similarly for two groups of sarcomas. In addition, transversion of G to C and A to T was detected at the K-ras gene in four of the seven sarcomas with the amplified c-myc gene, and these mutations are thought to be induced directly by methylcholanthrene. Thus, concomitant occurrence was observed for three seemingly unrelated mutations, amplification of the c-myc locus, point mutation of the K-ras gene, and instability at the hypervariable mouse minisatellite locus. The present study indicates a possible involvement of K-ras mutation and c-myc amplification in induction of genetic instability in methylcholanthrene-induced mouse sarcomas.

Genetic status of p53 and induction of apoptosis by radiation or isoflavones in human gastric carcinoma cell lines.

We have shown previously that various human cancer cell lines undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation or isoflavones. Here, we assessed the role of p53 gene in cell cycle and apoptosis following treatment of 11 gastric carcinoma cell lines with gamma-rays, genistein, biochanin A, or daidzein. Cell survival was measured by trypan blue staining, and apoptosis was assessed by fluorochrome staining. The rate of cell survival and apoptosis of the cells by gamma-irradiation or isoflavones did not correlate with p53 gene abnormalities. Flow cytometric measurement of DNA content demonstrated that while gamma-irradiation and genistein induced G(2) arrest, biochanin A and daidzein blocked the cell cycle of all carcinoma cells at G(1) phase. At multiple time points following irradiation, G(2) arrest was observed at 12-16 h in the wild-type and mutant p53 cell lines. Induction of p53 and p21 proteins was not observed in wild-type p53 lines after exposure to gamma-irradiation or isoflavones by Western blotting. Moreover, transfection of the wild-type p53 gene into MKN-1 cells failed to induce G(1) arrest by gamma-irradiation and genistein. Based on these results, we hypothesize that gastric cancer cells may possess a signal pathway which is different from the usual mechanisms of the p53-mediated DNA damage response in normal or hematopoietic tumor cells.

A new method for measuring cerebrospinal fluid flow in shunts.

An implantable device for measurement of cerebrospinal fluid (CSF) flow in a ventriculoperitoneal shunt tube has been developed. The unit is energized by an extracorporeal high-frequency generator (200 KHz), and electrolysis creates bubbles in the shunt tube. Velocity of bubble flow is detected by a pair of ultrasonic Doppler probes placed a certain distance apart on the skin surface and in parallel with the implanted tube. The CSF flow rate is calculated taking into account velocity and tube diameter, and is expressed in ml/min. The unit consists of a coil with a capacitor, a silicon diode to rectify the high frequency, and a Zener diode to regulate maximum output voltage of 20 V. The output is fed to a pair of platinum electrodes placed inside the unit's tunnel through which the CSF flows. These components are molded in epoxy resin and coated with medical-grade silicone rubber. In animal experiments, CSF flow rates ranging from 0.033 to 1.0 ml/min could be measured by this flowmeter. Clinically, CSF flow has been measured to date in several cases. In two cases of communicating hydrocephalus occurring after the onset of cerebrovascular disease, and in which the CSF flow was continuously monitored for 24 hours, the flow rate ranged between 0.05 and 0.78 ml/min. The CSF flow rate fluctuates in a 24-hour period, increasing in the morning, especially between 12 midnight and 6 a.m., which suggests a circadian rhythm.

Radiation-induced apoptotic cell death in human gastric epithelial tumour cells; correlation between mitotic death and apoptosis.

The mode of cell death in cells undergoing mitotic death after gamma-irradiation was studied in seven human gastric epithelial tumour cell lines and two strains of normal gastric fibroblasts. Apoptotic cells were frequently observed in all tumour lines after irradiation, whereas the two fibroblast strains were quite low in apoptosis frequency. The advent of apoptosis depended on the radiation doses and incubation time. Detailed analysis of one of the carcinoma lines, SH101-P4, revealed that G2-phase arrest was maximum at 12 h postirradiation. The cells began to escape G2 arrest by 24 h. Apoptotic cells began to increase at 12 h postirradiation and became maximal from 72 to 96 h. Apoptosis developed in the G1 phase of the cell cycle subsequent to the irradiation. These results suggest that apoptosis is one of the modes of mitotic death after irradiation.

Induction of a germline mutation at a hypervariable mouse minisatellite locus by 252Cf radiation.

Male C3H/HeN mice were exposed to 252Cf radiation and mated with unirradiated C57BL/6N females. F1 mice were analyzed for germline mutation at the paternally derived C3H/HeN allele of a hypervariable minisatellite locus, Ms6hm. This locus exhibited a high frequency of length change mutation spontaneously, and the mutation frequency of the paternally derived C3H/He allele in F1 mice born to unirradiated males was 8.4%. Exposure of male mice to 252Cf radiation resulted in even higher frequency of germline mutation. The spermatid stage germ cells were most sensitive to neutrons, and the mutation frequencies of the paternal allele were elevated to 18%, 26% and 24% for 0.35, 0.7 and 1.02 Gy of 252Cf radiation, respectively. Spermatozoa and spermatogonia stages were less sensitive and the mutation frequencies for 1.02 Gy of 252Cf radiation were 16% and 19%, respectively. The 252Cf radiation consisted of 35% gamma-rays and 65% neutrons. Assuming that these two radiations act additively, RBE of 252Cf neutrons for the induction of minisatellite mutation was calculated to be 5.9 for spermatozoa stage irradiation, 2.6 for spermatid stage irradiation and 6.5 for spermatogonia irradiation.

[A method for the continuos recording of intracraneal pressure]. / Metodo para registro continuo da pressão intracraniana

91 patients with acute head injuries, hydrocephalus, cerebral infarction, subarachnoid hemorrhage, encephalitis, intracerebral hemorrhage, or carbon monoxide intoxication have been so monitored by using the Numoto pressure switch by a method herein described. The main advantage has been the knowledge of the level of intracranial pressure at any given time and the early detection of a rising pressure when this phenomenon occurred. There were no complications except for 3 cases of infection. Two of these cases were minor purulent collections only at the site of exit of the tube in the scalp. One patient with a compound wound, cerebral laceration, and intracerebral hematoma developed a wound infection and brain abscess which required drainage.

A non-invasive CSF flowmeter.

A new non-invasive method for quantitative measurement of cerebrospinal fluid (CSF) flow in the ventriculo-peritoneal shunt tubing, used in hydrocephalus, has been developed. It is an implantable device which produces a bubble in the shunt tubing by electrolysis. This bubble is then detected in the tubing by an electrode arrangement using electric impedance or ultrasonically using a Doppler probe. The energy for electrolysis is supplied by extracorporeal high-frequency transmission. The CSF flow rate is calculated by the velocity of bubble flow in the tubing. CSF flow rates, ranging from 0.01 to 1.00 ml/min, have been measured in animal experiments with statistically good accuracy. In 11 clinical cases a flow range of between 0.01 and 1.93 ml/min have been observed.

[Intracranial pressure monitoring apparatus for clinical use balanced pressure sensors].

Three types of pressure sensors, (1) electric pressure switch, (2) fiber optic pressure switch and (3) pressure indicating bag for intracranial pressure monitoring which were developed by the author are described. Advantages and disadvantages between them are also discussed. The electric pressure switch is relatively simple in construction but has a possibility of producing micro-shock hazard in case of accidental electric leakage. The fiber optic pressure switch is the safest for the micro shock but its structure is rather complicated and fragile. The pressure indicating bag is simple to make and durable to use. However, it has a hydrostatic effect.

[CSF dynamics following shunt operation, with special reference to the diurnal changes of CSF circulation].

The purpose of this study is to study the pathophysiology of the cerebrospinal fluid (CSF) formation and circulation after a ventriculoperitoneal shunt operation. With the CSF flowmeter we developed, the CSF flow rate in the shunt tube has been measured non-traumatically over a 24-hour period in six patients. These include both communicating and noncommunicating hydrocephalus patients with ages ranging from 20 to 70. There were three cases of ruptured intracranial aneurysm, one cerebral contusion, one hypertensive brain stem hemorrhage and one occlusion of the aqueduct sylvius. Intraventricular pressure was continuously recorded for 24 hours prior to the shunt operation in each case, and the pressure changes were compared with the measured CSF flow rates in the shunt tube. The flow rate fluctuated between 0.05 ml/min and 1.2 ml/min with the supine position and high flow rates were detected in the early morning. Each case showed its own rhythm of CSF flow fluctuation during a 24-hour period, and the changes were compatible with the intraventricular pressure. It is suggested that there may be a relationship between these changes and an increased cerebral blood volume during the REM sleep stage.

[Cerebrospinal fluid absorption mechanism--based on measurement of CSF flow rate in shunt tube].

The cerebrospinal fluid (CSF) absorption mechanism in cases of hydrocephalus was investigated on the basis of measurements of CSF flow in a shunt tube after ventriculo-peritoneal shunt surgery, monitoring of intracranial pressure, CT findings, radioisotope cisternography, cerebral blood flow, EEG, PSP tests and changes in neurological findings. The subjects were 6 males and 7 females aged from 18 to 70. CSF flow rates in the shunt tubes were between 0.01 and 1.93 ml/min. Calculating the daily volume of CSF flow, the subjects were divided into two groups: Group A (8 patients) with a volume of less than 150 ml/day (0.01-0.25 ml/min), and Group B (5 patients) with between 150 and 500 ml/day (0.01-1.93 ml/min). Monitoring of intracranial pressure prior to the shunt operation was performed in 10 cases. These pressure values ranged between 4 and 25 mmHg (mean: 7-8 mmHg), and there was no difference between the two groups. The pre-and post-operative radioisotope cisternography findings indicated improvement of ventricular dilatation, periventricular lucency and ventricular reflux. After the shunt operations, there was neurological improvement in 6 of the 8 Group A cases but only in 2 of the 5 Group B cases. Considering the CSF flow volumes of the two groups, it appears that in Group A the shunt tube is not the main CSF circulation pathway. This could mean that resistance to CSF absorption in the cerebrospinal space has decreased after the shunt operation and there has been recovery of the physiological CSF absorption pathways. In other words, neurological improvement can be expected in this group A.

[A device to measure CSF flow in a shunt tube and its clinical application].

An implantable device for measurement of cerebrospinal fluid (CSF) flow in a shunt tube has been developed. The unit is energized by an extracorporeal high frequency generator (200 kHz), and electrolysis creates bubbles in the shunt tube. Bubble flow velocity is detected as reflected sound using a pair of ultrasonic Doppler probes (Saneisokkuki Doppler Flowmeter Type 1935) placed apart on the skin surface and in parallel with the tube. CSF flow is expressed in ml/min. by calculating velocity and tube diameter. The unit consists of a coil with a 200 kHz capacitor, a silicon diode to rectify the high frequency, and a Zener diode to regulate maximum output voltage of 20 V. The output is fed to a pair of platinum electrodes inside the unit's tunnel through which the CSF flows. The unit is moulded in epoxy resin and coated with medical grade silicon rubber. In vitro, CSF flow rates ranging from 0.033 ml/min to 1.0 ml/min. could be measured by this flowmeter model. In vivo, however, it was difficult to detect a flow rate of less than 0.006 ml/min. To measure the slower flow rate, a so-called bubble-detecting-tube made from an 11 cm stainless steel tube coated with silicon rubber is centrally inserted between the two ends of the separated shunt tube. The bubble flow velocity is detected by a tissue impedance detector's pair of probes placed apart on the skin surface. Clinically, CSF flow was measured in three cases of hydrocephalus (two cases of normal pressure hydrocephalus and one case of pineal tumor with non-cummunicating hydrocephalus). The flow rates were found to be, respectively, 0.10 ml/min., 0.063 ml/min., and 0.20 ml/min. The merits of the unit include its ability to repeatedly measure CSF flow at short intervals, and also to measure dynamic CSF flow under various conditions.

The same external signal differentially induced the c-myc expression in Burkitt lymphoma and B-lymphoblastoid cell lines.

An extracellular signal, such as phorbol-12-myristate-13-acetate (PMA), was found to reduce the c-myc expression in Burkitt lymphoma (BL) cells but augment the expression of the same gene in a B-lymphoblastoid cell line (B-LCL). Studies with Epstein-Barr virus (EBV)-converted BL cells demonstrated that the differential effect of PMA on c-myc expression was not due to alterations in the structure of the translocated c-myc gene, but to the difference in the intracellular milieu of the B cells. Experiments on the degradation rate in c-myc RNA suggested that this phenomenon in c-myc expression was exerted, at least in part, at the transcriptional level.