A two-site model for ApoB degradation in HepG2 cells.

Newly synthesized apolipoprotein B (apoB) undergoes rapid degradation in a pre-Golgi compartment in HepG2 cells. A major site of this early degradation seems to be on the cytosolic side of the endoplasmic reticulum (ER) membrane and is sensitive to N-acetyl-leucinyl-leucinyl-norleucinal (ALLN), which can inhibit neutral cysteine proteases and/or proteasome activity. Oleate (OA) treatment, which facilitates translocation of nascent apoB across the ER membrane, also reduces early degradation. In the present studies, we have used brefeldin A (BFA), which inhibits vesicular transport from the ER to the Golgi, to demonstrate that apoB can also be degraded by an ER luminal proteolytic activity that is distinct from the ALLN-sensitive proteases. Thus, when BFA-treated HepG2 cells were co-treated with ALLN, which protects apoB but does not facilitate its translocation into the ER lumen, degradation of newly synthesized apoB was significantly reduced compared with cells incubated with BFA alone. However, apoB degradation was rapid and complete when OA was added to media containing either BFA or ALLN/BFA. These results suggested that OA, by increasing translocation of nascent apoB into the ER lumen, exposed apoB to an ALLN-resistant proteolytic pathway. When we incubated HepG2 cells with dithiothreitol (DTT)/OA/BFA or DTT/OA/ALLN/BFA, degradation of apoB was inhibited. Furthermore, addition of DTT resulted in the accumulation of a 70-kDa amino-terminal fragment of apoB. Both full-length and amino-terminal apoB were degraded if DTT was removed from the incubation media; both were secreted if only BFA was removed. Thus, even after apoB is translocated into the ER lumen (thereby avoiding the initial proteolytic pathway), it can potentially be degraded by a lumenal proteolytic process that is ALLN-resistant but DTT-sensitive. The present results, together with previous studies, suggest that at least two distinct steps may be involved in the posttranslational degradation of apoB: 1) the first occurs while apoB is partially translocated and is ALLN-sensitive; and 2) the second occurs in the ER lumen and is DTT-sensitive. Finally, our results support the hypothesis that degradation of partially translocated apoB generates a 70-kDa amino-terminal fragment that is mainly degraded in the ER lumen by a DTT-sensitive pathway.

Fra(X)(q27.2), the common fragile site, observed in only one of 760 cases studied for the fragile X syndrome.

Cell cultures from 760 whole blood, amniotic fluid, chorionic villus sample, and peripheral umbilical blood sample specimens were exposed to multiple fra(X)(q27.3) induction systems (none had aphidicolin). Fifty-three exhibited the rare fragile site, fra(X)(q27.3) or FRAXA, none of which demonstrated the common fragile site or FRAXD at band Xq27.2. Only one cell in one of the negative whole blood FUdR-treated cultures from a mentally retarded male showed FRAXD. Therefore, it appears that FRAXD occurs very rarely in cultures treated to induce FRAXA since only one positive cell was observed in over 88,000 analyzed. It appears that very low frequencies of fra(X)(q27) can be accounted for only in part by the presence of the common fragile site since only one of 9 cases, each with one fra(X)(q27) positive cell, exhibited FRAXD and the others were FRAXA. After confirmation of FRAXA with direct DNA testing in a large number of low frequency cases, it should be possible to rely on the detection of very low frequencies of fra(X)(q27.3), e.g., 1% with at least 2 positive cells.

Cytogenetic and molecular analysis of human male germ cell tumors: chromosome 12 abnormalities and gene amplification.

We report karyotypic analysis of 24 male germ cell tumors (GCTs) with clonally abnormal karyotypes biopsied from testicular and extragonadal lesions from 20 patients belonging to the histologic categories seminoma, teratoma, embryonal carcinoma, choriocarcinoma, and endodermal sinus tumor. Chromosomes 1, 7, 9, 12, 17, 21, 22, and the X chromosome were nonrandomly gained in these tumors. Nonrandom structural changes affected most frequently chromosomes 1 and 12, the latter as i(12p) and/or del(12)(q13----q22). The i(12p) was seen in 90% of tumors which included all histologic subtypes and gonadal as well as extragonadal presentation. Our present results, along with those from published data on fresh GCT biopsies, establish that i(12p) is a highly nonrandom chromosome marker of all histologic as well as anatomic presentations of GCTs. in contrast, we found del(12)(q13----q22) exclusively in nonseminomatous GCTs (NSGCTs) and mixed GCTs (MGCTs) occurring in 44% of such lesions. Because successful cytogenetic analysis of fresh tumor specimens is not always possible, we developed a method based on DNA analysis to detect i(12p) as increased copy number of 12p. In addition to the changes affecting chromosome 12 identified above, we have detected, for the first time, cytological evidence of gene amplification in the form of homogeneously staining regions (HSRs) and double minute chromosomes (dmins) in treated as well as untreated primary extragonadal and metastatic GCTs and confirmed the presence of amplified DNA in one of these tumors at the molecular level by the in-gel renaturation method. Hybridization of DNA from cultured cells from an HSR-bearing tumor with a panel of probes for genes known to be amplified or otherwise perturbed in diverse tumor systems did not identify the amplified gene, suggesting amplification of a novel gene or genes. This study comprises the largest series of GCT cytogenetics attempted so far. Notably, it includes data on a series of primary mediastinal tumors, a group which previously has not been studied in any detail.

Progress toward an internal control system for fragile-X induction by 5-fluorodeoxyuridine in whole-blood cultures.

We have been attempting to develop a consistently reliable internal control to assure the effectiveness of the 5-fluorodeoxyuridine (FUdR) fragile-X [fra(X)] induction system. We carried out a systematic study of whole-blood specimens cultured from 56 individuals from two different laboratories. An analysis of nearly 9,000 cells demonstrated: (1) the importance of establishing baseline levels of fragile sites in each laboratory, and (2) that a combination of common fragile sites (different for each laboratory) could serve as a consistently reliable indicator of the effectiveness of the FUdR fra(X) induction system. It was suggested that a non-FUdR culture(s) should be incorporated into a laboratory's fra(X)-screening protocol, so that if there are any doubts about the effectiveness of the FUdR system a comparison to background or spontaneously occurring fragile sites can be made within the laboratory. Repeat cultures are recommended where no increase in common fragile-site frequency is observed in the FUdR induction system, and where fra(X) was strongly suspected but not found. In addition, the necessity of using more than one fra(X) induction system in whole-blood cultures was demonstrated, including the effectiveness of an FUdR/excess thymidine double-induction system. Finally, 2 cases of apparent mosaicism for Klinefelter syndrome in fra(X) individuals were observed.

Study of spindle microtubule reassembly in cells from Alzheimer and Down syndrome patients following exposure to colcemid.

Numerous intraneuronal neurofibrillary tangles and senile (neuritic) plaques are the two characteristic lesions in Alzheimer disease (AD) and adult Down syndrome (DS). Evidence indicates that microtubule assembly is impaired in AD. We studied spindle microtubule repolymerization rates in EBV-transformed lymphoblasts from AD, DS, and control individuals after colcemid exposure. The distinctive arrangement of microtubules in spindle and its size make this structure an obvious choice for study. Recovery trends in the three patient groups differed significantly; in particular, the controls showed an earlier appearance of intact spindle microtubules than AD. Other researchers found similar results using AD fibroblasts. The results from the DS cells were inconsistent and difficult to interpret. It is unclear how the AD microtubules differ from controls, or whether a relationship exists between the altered microtubule repolymerization kinetics observed in this study and the presence of neurofibrillary tangles in AD patients. A difference in repolymerization rates can be the basis for a diagnostic test for AD if it can be verified.

Recent experience in prenatal fra(X) detection.

At least 35 cases of prenatal fra(X) diagnosis have been confirmed and reported. Amniotic fluid, fetal blood and chorion -ic villus samples have exhibited fra(X) (q27.3) in cultures from 26 males and 9 females. Here we have detected fra(X) in female and male amniotic fluid specimens, AF1/fra(X),X and AF2/fra(X),Y, respectively, and a male CVS/fra(X),Y using both FUdR and excess thymidine (THY) to demonstrate the marker chromosome. Both FUdR and THY detected fra(X) and usually FUdR was superior to THY with the exception of placental cultures. It was important to examine more than one culture per protocol since no fra(X) was observed in one AF2 FUdR culture while another exhibited 19.2% expression. Similarly, confirmation studies in lung fibroblast cultures for AF2 exhibited 4.3% fra(X) in one lab while another found negative results. A similar observation in whole blood cultures was also made recently by us. In addition, we have recently experienced our first false negative fra(X),X prenatal diagnosis. We have observed another case where only one cell in 300 exhibited fra(X) where the male fetus was 50% at-risk and was referred to us after the 20th week of gestation by sonography. On the basis of our experience we recommend the following: 1) the excess THY fra(X) induction system is effective but not superior to FUdR; 2) at least two duplicate cultures per induction system should be analyzed for the marker chromosome to avoid the possibility of false-negative diagnosis; 3) where fra(X) is not demonstrated or is present in very low frequencies in CVS and/or amniotic fluid cultures, complementary DNA marker studies and/or fetal blood cultures must be made available; 4) gestational age dating by ultrasonography is recommended as early as possible.

Constitutive fragile sites in fra(X) individuals.

Recently, it was proposed that the constitutive fragile site at 3p14 be used as an "internal control" to indicate the effectiveness of the FUdR fragile site induction system. We have tested this hypothesis by determining the frequency of constitutive fragile sites at 1p31, 3p14, and 16q23 in cultures from 42 known fra(X) individuals. At least 50 cells were analyzed from each case. Seventy-four percent (31/42), 95% (40/42) and 90% (38/42) of the fra(X) individuals exhibited frequencies of less than 4% at constitutive fragile sites 3p14, 1p31 and 16q23, respectively. Of the 42 individuals tested, 12 or 28.6% showed no fragility at any of the 3 sites studied. On the other hand, at least one constitutive fragile site was observed in 50 cells studied from over 70% of the 42 people studied. It is suggested that "positive controls" continue to be used, while at the same time recording all fragile sites to identify a combination of constitutive fragile sites that may serve as an internal control indicator, and that DNA marker studies be used to complement cytogenetic testing.

Fragile X expression increased by low cell-culture density.

The effect of cell density on expression of fra(X) was studied. A lymphoblast cell line from one fra(X) individual and whole blood from another individual was tested at various cell densities using RPMI-1640 with FUdR (0.1 microM) 24 hrs before harvest. Densities from 0.25 X 10(5) to 2 X 10(6) cells/ml were tested. Chromosomes were G-banded and analyzed for fra(X) frequency. Increased density caused fra(X) frequency to decline in lymphoblasts and whole blood. In the established line low density fra(X) frequency was 51.2% and decreased to 6.5% at the high density. In blood fra(X) frequency was 34.7% at low density and decreased to 18% fra(X) in high density. We suspect that decay of the FUdR effect may explain the results. Our results suggest that to maximize fra(X) frequency, cultures should be inititated at low density. This may be important in analysis of low-percentage fra(X) patients, nonexpressing female carriers, and obligate nonpenetrant males.

Low frequencies of apparently fragile X chromosomes in normal control cultures: a possible explanation.

Low frequencies of apparently fragile X [fra(X)] chromosomes have been reported in normal control, short-term, whole blood cultures, and they have been noted in both amniocyte and fetal blood cultures. However, there is currently no universal agreement on the lowest frequency for fra(X)(q27) that is diagnostic for the fragile X syndrome. Here, we present our observations on low levels of apparently fra(X) chromosomes in normal samples. We observed frequencies of 0.5% in short-term whole blood cultures and 0.9% in amniotic fluid cell cultures. In 1982, Steinbach et al. described nonspecific telomeric structural changes (TSC) and suggested that such low frequencies of apparently fra(X) chromosomes in normal material may be occurring by the same mechanism that is responsible for TSC formation. To determine if TSC formation can explain the significant baseline frequencies of fra(X) in normal controls, 10,457 cells were screened from 178 individuals referred for fra(X) analysis. Our findings indicated that TSC are not randomly distributed across chromosomes but tend to occur at specific sites. Based on our observations, we offer the hypothesis that the low frequency of apparent fra(X) in normal individuals may be due to nonrandom TSC distribution.

Fragile X chromosome frequency is consistent temporally and within replicate cultures.

Fragile X frequencies differ widely between different fra(X)-positive individuals. The basis for this variation is unknown, but may reflect genetic differences or unknown environmental factors. To determine more fully the individual and familial variation in fra(X) frequencies, we studied 15 individuals. The present study showed that fra(X) frequency, with few exceptions, was consistent for the same individual both over time and within replicate cultures. This confirms previous observations by others on the consistency of fra(X) expression by others, and indicates the extent of expected variability within replicate cultures. Consideration of this variability should enable improved fra(X) identification.

Cytogenetic studies of hairy cell leukemia.

Mononuclear cells from peripheral blood and spleen of four patients with hairy cell leukemia (HCL) were cultured for 1 to 10 days with and without phytohemagglutinin (PHA) stimulation. Blastogenesis and mitoses were not observed in the unstimulated cells cultured for up to 3 days. Prolonging the culture time of unstimulated cells to 7 days produced blastogenic response; a further increase in culture time to 10 days showed transformed cells but not mitoses. Poor PHA response was noted in the cultures stimulated for 3 days, but cells cultured for 7 days and then stimulated with PHA entered mitosis. Surface immunoglobulins were exhibited in 78-94% of the cells in the blood and spleen of patients. In addition, these cells had tartarate-resistant acid phosphatase and were able to phagocytize latex particles by which properties they were identified as hairy cell leukemia cells. Chromosome abnormalities were present in a proportion of cells of each patient, none of them, however, were clonal.

A simple in-vitro test for assessing the sensitivity of lymphocytes to chlorambucil.

The sensitivity of lymphocytes to chlorambucil has been assessed by a simple in-vitro test which has been applied to the cells of normal controls and of patients with chronic lymphocytic leukaemia. The degree of sensitivity varied amongst the normal controls and in-vitro resistance of the lymphocytes in the patients was sometimes found in the absence of in-vivo experience of the drug. Resistance in-vitro tended to be associated with very high total peripheral blood lymphocyte counts but not with the age of the patient. Where the information was available the in-vitro sensitivity test agreed with the results of biochemical estimations of drug resistance and with the clinical responses to the drug. It is suggested that this test may have applications in patient management.