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Abstract Background Supernumerary marker chromosomes (SMCs) are structurally abnormal extra chromosomes that cannot be unambiguously identified by conventional banding techniques. In the past, SMCs have been characterized using a variety of different molecular cytogenetic techniques. Although these techniques can sometimes identify the chromosome of origin of SMCs, they are cumbersome to perform and are not available in many clinical cytogenetic laboratories. Furthermore, they cannot precisely determine the region or breakpoints of the chromosome(s) involved. In this study, we describe four patients who possess one or more SMCs (a total of eight SMCs in all four patients) that were characterized by microarray comparative genomic hybridization (array CGH). Results In at least one SMC from all four patients, array CGH uncovered unexpected complexity, in the form of complex rearrangements, that could have gone undetected using other molecular cytogenetic techniques. Although array CGH accurately defined the chromosome content of all but two minute SMCs, fluorescence in situ hybridization was necessary to determine the structure of the markers. Conclusion The increasing use of array CGH in clinical cytogenetic laboratories will provide an efficient method for more comprehensive characterization of SMCs. Improved SMC characterization, facilitated by array CGH, will allow for more accurate SMC/phenotype correlation.

Abstract Background X chromosome inactivation (XCI) is that aspect of mammalian dosage compensation that brings about equivalence of X-linked gene expression between females and males by inactivating one of the two X chromosomes (Xi) in normal female cells, leaving them with a single active X (Xa) as in male cells. In cells with more than two X's, but a diploid autosomal complement, all X's but one, Xa, are inactivated. This phenomenon is commonly thought to suggest 1) that normal development requires a ratio of one Xa per diploid autosomal set, and 2) that an early event in XCI is the marking of one X to be active, with remaining X's becoming inactivated by default. Results Triploids provide a test of these ideas because the ratio of one Xa per diploid autosomal set cannot be achieved, yet this abnormal ratio should not necessarily affect the one-Xa choice mechanism for XCI. Previous studies of XCI patterns in murine triploids support the single-Xa model, but human triploids mostly have two-Xa cells, whether they are XXX or XXY. The XCI patterns we observe in fibroblast cultures from different XXX human triploids suggest that the two-Xa pattern of XCI is selected for, and may have resulted from rare segregation errors or Xi reactivation. Conclusion The initial X inactivation pattern in human triploids, therefore, is likely to resemble the pattern that predominates in murine triploids, i.e., a single Xa, with the remaining X's inactive. Furthermore, our studies of XIST RNA accumulation and promoter methylation suggest that the basic features of XCI are normal in triploids despite the abnormal X:autosome ratio.

Loss-of-function mutations in the WRN helicase gene cause Werner syndrome- a progeroid syndrome with an elevated risk of cancer and other age-associated diseases. Large numbers of single nucleotide polymorphisms have been identified in WRN. We report here the organismal, cellular, and molecular phenotypes of variant rs3087425 (c. 2500C > T) that results in an arginine to cysteine substitution at residue 834 (R834C) and up to 90% reduction of WRN helicase activity. This variant is present at a high (5%) frequency in Mexico, where we identified 153 heterozygous and three homozygous individuals among 3,130 genotyped subjects. Family studies of probands identified ten additional TT homozygotes. Biochemical analysis of WRN protein purified from TT lymphoblast cell lines confirmed that the R834C substitution strongly and selectively reduces WRN helicase, but not exonuclease activity. Replication track analyses showed reduced replication fork progression in some homozygous cells following DNA replication stress. Among the thirteen TT homozygotes, we identified a previously unreported and statistically significant gender bias in favor of males (p = 0.0016), but none of the clinical findings associated with Werner syndrome. Our results indicate that WRN helicase activity alone is not rate-limiting for the development of clinical WS.

This trial tested the safety and efficacy of a regimen consisting of hydroxyurea followed by azacitidine, 75 mg/m(2) for 7 days, and gemtuzumab ozogamicin, 3 mg/m(2) on day 8, in older patients with newly diagnosed acute myeloid leukemia. Those achieving a complete remission received 1 consolidation treatment followed by 4 cycles of azacitidine. The patients were stratified into good-risk (age 60-69 years or performance status 0-1) and poor-risk (age ≥70 years and performance status 2 or 3) groups. Specific efficacy and safety goals were defined as being supportive of further study of the regimen. Eighty-three patients were registered in the good-risk cohort and 59 in poor-risk cohort, with median age of 71 and 75 years, respectively. In the good-risk group, 35 patients (44%) achieved a complete remission. Median relapse-free and overall survivals were 8 and 11 months, respectively. Six patients (8%) died within 30 days of registration. In the poor-risk group, 19 (35%) achieved a complete remission. Median relapse-free and overall survivals were 7 and 11 months, respectively. Seven patients (14%) died early. The results of this trial met predefined goals for efficacy and safety for the poor-risk cohort but not the good-risk group. .

Background: Proteoglycans, a complex group of extracellular matrix (ECM) molecules, are elevated in benign prostatic hyperplasia (BPH). Versican is a stromal proteoglycan present in prostate tissue. Versican expression is elevated in tissues with increased proliferation. Based on these observations, we determined the extent and distribution of versican expression in prostates with BPH., Methods: The involvement of versican in BPH nodules was compared with levels in non-nodular transition (TZ) and peripheral zone (PZ) tissues from 18 human prostate glands using immunohistochemistry, Northern blots and/or QRTPCR to localize versican and quantify versican mRNA transcript levels, and Western blots to assess gene product levels., Results: Increased versican immunoreactivity was observed in the stroma of BPH nodules. Higher steady state levels of versican variants V0, V1, and V3 mRNA transcript and gene product were detected in the nodular tissues than in the non-nodular TZ or PZ parenchyma., Conclusions: These results suggest that versican may play a role in nodule formation in BPH.

Mammalian DNA polymerase delta (Pol delta) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol delta. Homozygous Pold1(L604G/L604G) and Pold1(L604K/L604K) mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1(+/L604G) mice did not differ, while that of Pold1(+/L604K) mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1(+/L604K) mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase delta reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer.

Background: X chromosome inactivation (XCI) is that aspect of mammalian dosage compensation that brings about equivalence of X-linked gene expression between females and males by inactivating one of the two X chromosomes (Xi) in normal female cells, leaving them with a single active X (Xa) as in male cells. In cells with more than two X's, but a diploid autosomal complement, all X's but one, Xa, are inactivated. This phenomenon is commonly thought to suggest 1) that normal development requires a ratio of one Xa per diploid autosomal set, and 2) that an early event in XCI is the marking of one X to be active, with remaining X's becoming inactivated by default., Results: Triploids provide a test of these ideas because the ratio of one Xa per diploid autosomal set cannot be achieved, yet this abnormal ratio should not necessarily affect the one-Xa choice mechanism for XCI. Previous studies of XCI patterns in murine triploids support the single-Xa model, but human triploids mostly have two-Xa cells, whether they are XXX or XXY. The XCI patterns we observe in fibroblast cultures from different XXX human triploids suggest that the two-Xa pattern of XCI is selected for, and may have resulted from rare segregation errors or Xi reactivation., Conclusion: The initial X inactivation pattern in human triploids, therefore, is likely to resemble the pattern that predominates in murine triploids, i.e., a single Xa, with the remaining X's inactive. Furthermore, our studies of XIST RNA accumulation and promoter methylation suggest that the basic features of XCI are normal in triploids despite the abnormal X:autosome ratio.

The ability of 4-color flow cytometry (FC) to help identify myelodysplastic syndromes (MDSs) was evaluated in 124 bone marrow aspirates from unselected patients with unexplained cytopenias and/or monocytosis. The morphologic features of bone marrow aspirate smears were correlated with FC and cytogenetic findings blindly, and patterns of antigen expression were compared with patterns seen in nonneoplastic and normal marrow specimens. Of 124 cases, 58 (46.7%) had definitive FC abnormalities ("flow-abnormal"), 19 cases (15.3%) had mild FC abnormalities of indeterminate significance, and 47 cases (37.9%) had essentially normal FC. Highly significant differences were identified between the flow-abnormal group and other groups in mean myeloid blast percentages and numbers of abnormal antigens expressed, even when the analysis was limited to cases with fewer than 5% myeloid blasts. Strikingly, flow-abnormal cases constituted 50 (89%) of the 56 morphologically abnormal cases and 31 (94%) of the 33 cytogenetically abnormal cases, demonstrating the strong concordance of FC-identified antigenic abnormalities with morphologic features and cytogenetics in the evaluation of patients with unexplained cytopenias.

Inflammatory leiomyosarcoma, a rare entity first described in 1995, has been characterized by smooth muscle differentiation, a near-haploid karyotype, and a surprisingly good prognosis. The morphology is similar to that of conventional leiomyosarcoma admixed with a chronic inflammatory infiltrate. Thus far, only 15 cases have been reported in the English language literature. We report the clinical and pathological features of 3 additional cases of inflammatory leiomyosarcoma. Two women (ages 64 and 25, respectively) and 1 man (age 32) presented with a thigh, ovary, and lung mass, respectively. Inflammatory symptoms, such as anorexia, fever, night sweats, abdominal pain, and diarrhea, coincided with the thigh and ovarian primaries. Immunohistochemical studies revealed diffuse positivity for desmin and poor expression for other smooth muscle and skeletal muscle markers (muscle-specific actin [0/3], alpha-smooth muscle actin 1/3 [focal], calponin [1/3], caldesmon [0/3], and myogenin [0/3]). CD68 was diffusely positive in both the histiocytes and spindle cell component in all cases. Ultrastructural evaluation of 1 case (lung primary) lacked definitive smooth muscle differentiation. Cytogenetic analysis in 1 of 2 cases that were karyotyped, identified a near-haploid karyotype, which has been reported in other cases of inflammatory leiomyosarcoma. The other case showed 2 clonal populations of cells with interstitial deletions of the short arm of chromosome 8 and the long arm of chromosome 9, respectively. The case without cytogenetic data was intimately associated with an ovarian mature teratoma. These data also suggest that inflammatory leiomyosarcoma may lack smooth muscle differentiation, characterized by diffuse immunoreactivity for desmin but lack of immunoreactivity for alpha-smooth muscle actin, calponin, and caldesmon. In addition, 2 of the 3 cases developed distant metastases to the lungs, which suggests that these lesions may have a worse prognosis than previously believed.

Background: Intramuscular lipomas and atypical lipomatous tumors (ALT) are common deep-seated lipomatous tumors of the chest wall and extremities. Distinguishing between these two entities can be difficult based on histologic analysis alone. However, the cytogenetic profiles of ALT and intramuscular lipomas are distinct. Correct classification is important, because aggressive local disease recurrence occurs more frequently in patients with ALT than in patients with intramuscular lipoma. The authors examined their single institutional experience and correlated their classification with clinical features and outcome., Methods: In the current study, 106 patients with deep-seated, well differentiated adipose tumors of the chest wall and extremities were classified as having ALT or intramuscular lipoma using a combined approach of histology and cytogenetics, if available. The classification was correlated with clinicopathologic features and follow-up data., Results: Fifty-five patients were classified as having intramuscular lipoma and 51 were classified as having ALT. Classification did not correlate with age and gender (P = 0.28 and P = 0.96, respectively). Intramuscular lipomas were smaller than ALTs (P < 0.0001), but there was significant overlap between the 2 groups. ALT occurred preferentially in the lower extremity (P < 0.0009). Four percent of patients with intramuscular lipomas and 27% of patients with ALTs developed local disease recurrence (P = 0.0006). Disease recurrence did not correlate with patient age at diagnosis, patient gender, tumor size, and tumor location (P = 0.45, P = 0.26, P = 0.49, and P = 0.28, respectively). Within the subset of patients with ALTs, disease recurrence did not correlate with patient age at diagnosis, patient gender, or tumor location (P = 0.38, P = 0.54, and P = 0.86, respectively)., Conclusions: Classification of deep-seated, well differentiated lipomatous tumors of the extremities and chest wall using a combined approach of histology and cytogenetics correlated well with biologic behavior/disease recurrence. This combined approach is advocated to better stratify patients for treatment purposes and follow-up., ((c) 2004 American Cancer Society.)

In a 5-year survey of nonpromyelocytic/nonmonocytic acute myeloid leukemias (AMLs) diagnosed in the University of Washington Hematopathology Laboratory, we identified 19 cases containing distinctive, cup-like nuclear indentation in 10% or more of the blasts ('AML-cuplike'). Fourteen of these cases (74%) demonstrated near-complete loss of HLA-DR expression, while the other five cases showed partial loss of HLA-DR. A total of 16 of the cases (84%) demonstrated internal tandem duplication (ITD) of the Flt3 gene. When compared to a selected set of AMLs lacking this nuclear morphology, AML-cuplike was significantly more likely to lack HLA-DR and CD34 expression, to express CD123 without CD133, to have a normal karyotype, and to harbor the Flt3 ITD. To characterize AML-cuplike in an unselected series of AMLs, we analyzed 42 consecutive nonpromyelocytic/nonmonocytic AMLs diagnosed in our laboratory during a 6-month period in 2002. Strikingly, in this unselected series, there was a statistically significant coincidence of invaginated nuclear morphology, loss of HLA-DR, and presence of the Flt3 ITD beyond that expected if these three features were unrelated, suggesting that AMLs with these three features may represent a distinct AML subset.

Human diploid fibroblasts (HDF) rarely, if ever, undergo spontaneous transformation to an immortalized cell type. Here we report the immortalization of an HDF cell line following transduction with cyclin A2 or cdk1 human genes via retroviral vectors. Fluorescence in situ hybridization (FISH) studies using the retroviral vector as a probe indicate that these cell lines are monoclonal. No telomerase activity could be detected in these cell lines, and the telomere length in the immortalized cells was observed to be 10-20 kb longer than that in low-passage cells from the parental fibroblast line. Cytogenetic studies revealed that the immortal lines share common chromosomal aberrations. FISH studies with a probe for p53 revealed loss of one copy of this gene which was associated with reduced steady-state levels of both p53 and p53-regulated p21(WAF1/Sdi1/CIP1) messages in both quiescent and proliferating immortalized cultures relative to the parental cells. Additional FISH studies with probes for p16(INK4a) and Rb, carried out after the immortalized cells proliferated in excess of 100 population doublings, also revealed loss of one copy of these genes in both cell lines. These cell lines, together with the well-characterized parental cells, could provide useful research material for the study of the mechanisms of immortalization and of regulation of proliferative senescence in HDF.

As there has been no previous information on the consequences of telomerase expression in genetically altered, mortal cells derived from pre-malignant tissue, we sought to determine the effect of hTERT (human catalytic subunit of telomerase reverse transcriptase) transduction of pre-malignant cell strains from Barrett's esophagus that do not contain telomerase activity and possess a finite lifespan. Primary cultures of Barrett's esophageal epithelium transduced with a retrovirus containing hTERT were characterized by growth factor requirements, cytogenetics and flow cytometry. Expression of telomerase lengthened telomeres and greatly extended the lifespan of hTERT transduced (hTERT+) Barrett's esophagus cells. Growth factor dependency of the hTERT+ cultures remained largely similar to the parental cultures, although there was a modest increase in the ability to grow in agar. Chromosomal instability, measured by both karyotypic and FISH (fluorescence in situ hybridization) analyses, was reduced but not abrogated by hTERT transduction, suggesting that telomerase expression can enhance genomic stability. However, the persistence of residual instability gave rise to new clonal and non-clonal genetic variants, and in one hTERT+ culture a new DNA aneuploid population was observed, the only time such a ploidy shift has been seen in Barrett's cell strains in vitro. These in vitro observations are analogous to the clinical progression to aneuploidy that often precedes cancer in Barrett's esophagus, and suggest that reactivation of telomerase may be permissive for continued genetic evolution to cancer. Long-lived Barrett's esophagus epithelial cultures should provide a useful in vitro model for studies of neoplastic evolution and chemopreventive therapies.

Myelolipomas are benign tumors composed of both mature adipose and myeloid tissues. They typically present as an incidental mass in one of the adrenal glands proper. However, they can occur in ectopic adrenal tissue or, rarely, without associated adrenal tissue in various locations and can grow to weights of several kilograms. These tumors have been linked to endocrinopathies, such as Cushing disease and congenital adrenal hyperplasia, which involve overproduction of adrenocorticotropic hormone. We report a case of three giant adrenal myelolipomas arising in a persistently virilized female with congenital adrenal hyperplasia, supporting a role for hormonal stimuli in myelolipoma formation.

Complete remission and long-term survival rates are low for older adults treated for acute myeloid leukemia (AML). Because of favorable phase 2 data using mitoxantrone and etoposide, we conducted a phase 3 study (SWOG-9333) in which patients over 55 years of age with previously untreated AML were randomized to receive mitoxantrone (10 mg/m(2) per day x 5) and etoposide (100 mg/m(2) per day x 5) [ME], or cytarabine (200 mg/m(2) per day x 7) and daunorubicin (45 mg/m(2) per day x 3) [AD] as induction therapy. The randomization was stratified by age, onset of leukemia, and multidrug resistance phenotype. Over a 4-year period, 328 eligible patients from 66 institutions were enrolled. The complete remission rate was 34% (95% confidence interval [CI] 26%-41%) for patients in the ME and 43% (CI 35%-51%) for patients in the AD treatment arm (one-tailed P value.96). The rates of resistant disease were 43% (CI 35%-51%) and 34% (CI 27%-42%), respectively, for the 2 treatment arms (one-tailed P value.95). The estimated overall survival at 2 years was 11% (CI 6%-15%) and 19% (CI 12%-25%) for patients randomized to ME and to AD induction therapy, respectively (one-tailed P value.99). After accounting for the independent prognostic factors associated with survival (karyotype, performance status, age, white blood cell count), exploratory analysis suggested there was a worse survival for patients who received ME compared with AD induction therapy (2-tailed P value.0066). We conclude that the results of our study do not demonstrate any benefit to the use of ME induction chemotherapy instead of AD in older patients with AML.

Purpose: Dermatofibrosarcoma protuberans is caused by activation of the platelet-derived growth factor B (PDGFB) receptor, a transmembrane tyrosine kinase. We investigated the response of dermatofibrosarcoma protuberans to the tyrosine kinase inhibitor imatinib mesylate., Patients and Methods: A patient with unresectable, metastatic dermatofibrosarcoma protuberans received imatinib mesylate (400 mg bid). Response to therapy was assessed by [18F]fluorodeoxyglucose (FDG) positron emission tomography, magnetic resonance imaging, and histopathologic and immunohistochemical evaluation., Results: The patient was treated for 4 months with imatinib mesylate. The hypermetabolic uptake of FDG fell to background levels within 2 weeks of treatment, and the tumor volume shrank by over 75% during the 4 months of therapy, allowing for resection of the mass. There was no residual viable tumor in the resected specimen, indicating a complete histologic response to treatment with imatinib mesylate., Conclusion: Imatinib mesylate is highly active in dermatofibrosarcoma protuberans. The dramatic response seen in this patient demonstrates that inhibition of PDGFB receptor tyrosine kinase activity can significantly impact viability of at least one type of solid tumor.

The chromosome abnormalities observed in a dedifferentiated chondrosarcoma are reported. A new molecular cytogenetic technique, spectral karyotyping, was used to identify and confirm structural rearrangements in this case. A review of the literature revealed that nine cases have been reported, in eight of which a complete description of the cytogenetic abnormalities was described. Structural aberrations were most frequently reported in chromosomes 1 and 9, and chromosomes 7 and 19 were most frequently observed to be involved in numerical aberrations (trisomy and tetrasomy). In chondrosarcomas, structural aberrations in chromosomes 1 and 9 and trisomy or tetrasomy of chromosome 7 are among the more frequently observed aberrations.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative disorder in which recurrent abnormalities of chromosome 20 have been reported. We report the case of a 76-year-old woman with CNL with partial deletion of the long arm of chromosome 20 in a subset of bone marrow metaphases, suggesting coexistence of a clonal stem cell disorder and normal hematopoiesis. Review of the literature suggests that such mosaicism is common in CNL, possibly accounting for the favorable prognosis observed in many patients with this disorder., (Copyright 2000 Wiley-Liss, Inc.)

Cyclin E-Cdk2 is an evolutionary conserved cyclin-dependent kinase (CDK) complex that drives the G1 to S phase transition of the cell cycle. A novel cDNA encoding a HECT family protein also containing RCC1-like repeats was isolated by a yeast two-hybrid screening using both cyclin E and its inhibitor p21. The protein product of this cDNA, Ceb1, interacts with various cyclin subunits of CDKs in mammalian cells. Expression of Ceb1 is specifically detected in testis and ovary and is highly elevated when the functions of the tumor suppressor proteins, p53 and RB, are compromised by mutations or viral oncoproteins. The present results suggest that Ceb1 may play a critical role when its expression and the CDK activity are upregulated by inactivation of p53 and RB., (Copyright 1999 Academic Press.)

Cytoplasmic extracts from early-passage (young), late-passage (senescent) normal human fibroblast (HF) cultures and immortalized human cell lines (HeLa, HT-1080, and MANCA) were analyzed for their ability to support semiconservative DNA synthesis in an in vitro SV40-ori DNA replication system. Unsupplemented extracts from the three permanent cell lines were demonstrated to be active in this system; whereas young HF extracts were observed to be minimally active, and no activity could be detected in the senescent HF extracts. The activity of these extracts was compared after supplementation with three recombinant human replication factors: (1) the catalytic subunit of DNA polymerase alpha (DNA pol-alpha-cat), (2) the three subunits of replication protein A (RPA), and (3) DNA topoisomerase I (Topo I). The addition of all three recombinant proteins is required for optimum activity in the young and senescent HF extracts; the order of the level of activity is: transformed > young HF > senescent HF. Young HF extracts supplemented with RPA alone are able to support significant replicative activity but not senescent extracts which require both RPA and DNA pol-alpha-cat for any detectable activity. The necessary requirement for these factors is confirmed by the failure of unsupplemented young and senescent extracts to activate MANCA extracts that have been immunodepleted of DNA pol-alpha-cat or RPA. Immunocytochemical studies revealed that RPA, DNA pol-alpha, PCNA, and topo I levels are higher in the immortal cell types used in these studies. In the HF cells, levels of DNA pol-alpha-cat and PCNA are higher (per mg protein) in the low-passage than in the senescent cells. By contrast, RPA levels, as determined by immunocytochemical or Western blot studies, were observed to be similar in both young and senescent cell nuclei. Taken together, these results indicate that the low to undetectable activity of young HF extracts in this system is due mainly to reduced intracellular levels of RPA, while the senescent HF extracts are relatively deficient in DNA polymerase alpha and probably some other essential replication factors, as well as RPA. Moreover, the retention of RPA in the senescent HF nuclei contributes to the low level of this factor in the cytoplasmic extracts from these cells.

Cementum-derived growth factor (CGF) is a 14 kDa polypeptide sequestered in tooth cementum. It is an IGF-I like molecule that is weakly mitogenic to fibroblasts, but its mitogenic action is synergistically potentiated in the presence of epidermal growth factor (EGF) or serum. We have examined whether the CGF affects cyclin E levels and the activity of cyclin-dependent kinase (Cdk) associated with this cyclin, and whether these changes contribute to the synergism in mitogenic activity between CGF and EGF. Optimal DNA synthesis by serum-starved human gingival fibroblasts required the presence of CGF for 0-12 h and EGF for 0-3 h. Therefore, cells were serum starved for 48 h and then exposed to CGF, EGF, or CGF + EGF. Cells incubated with 10% fetal bovine serum (FBS) served as positive controls. At various time points after the addition of growth factors, cyclin E levels were examined by Western analysis. Cdk associated with cyclin E was immunoprecipitated with anti-cyclin E antibody and kinase activity was measured using H1 histone as substrate. Cyclin E and the H1 kinase activity levels increased after 8-12 h in cells exposed to CGF and in positive controls exposed to 10% FBS. They returned to basal level 4 h later in cells exposed to CGF alone, whereas in the presence of CGF + EGF and FBS they remained elevated for up to 20 h. The cyclin E levels did not increase in the presence of EGF alone. Cyclin-dependent kinase inhibitors p21cip1 and p27kip1 were barely detectable in these cells. Fibroblasts transfected with LXSN-cyclin E, a retroviral vector containing cyclin E cDNA, overexpressed cyclin E and their steady-state cyclin E-Cdk activity was higher than control cells. DNA synthesis by cyclin E overexpressing cells was higher, but optimal DNA synthesis by these cells required the presence of CGF and EGF. These results show that CGF action involves an increase in the levels of cyclin E and E-Cdk activity and that the higher levels are maintained in the presence of both CGF and EGF. They also indicate that sustained high cyclin E levels and Cdk2 activity during G1 phase are necessary, but not sufficient, for optimal mitogenic response in human fibroblasts.

A de novo tandem inverted duplication of 10p was diagnosed in a 17-week fetus. The appearance of GTG banded preparations and the results of fluorescence in situ hybridization (FISH) studies are consistent with duplication of the entire arm, including the telomere. The FISH studies also demonstrated the presence of chromosome 10 alphoid repeats at the junction between the inverted segment and the long arm, consistent with the presence of the entire long arm of the abnormal chromosome. Therefore, this is a case of pure trisomy 10p without an associated deficiency of any other chromosome segment. A comparison of the phenotype associated with pure trisomy 10p and trisomy associated with a duplication/deficiency state documented a higher frequency (of borderline significance) of clubfoot and high-arched/cleft palate in the cases of pure trisomy. The frequency of palatal anomalies was observed to be significantly higher in the cases where the breakpoint of the trisomic segment is in the most proximal band (10p11). However, other clinical manifestations were observed inconsistently, even in the cases with pure, nearly complete trisomy 10p. Therefore, a clearly defined trisomy 10p clinical syndrome could not be documented in this study.

There is now evidence that the immediate cause of the loss of proliferative capacity in senescent cells is mediated by a specific inhibitor. If this tentative interpretation is correct, the next hurdle will be to determine mechanism(s) that regulate this putative senescence cell inhibitor that would, in effect, be the determinant of proliferative life span. One previously proposed hypothesis predicts that the decline of replicative activity is analogous to a checkpoint response to accumulated chromosomal damage (Rosenberger et al., 1991). Advances in our basic understanding of the nature of DNA damage, DNA repair mechanisms, and the response of eukaryotic cells to accumulated DNA damage provide a solid rationale for a reassessment of the causal role of the accumulation of chromosomal damage in cell senescence in vitro.

We describe 2 cases of balanced reciprocal translocation (BRT) mosaicism. The frequency of this aberration in the population referred to our laboratory was determined and compared to those frequencies reported in the literature by other clinical cytogenetics laboratories. The extent of BRT mosaicism was also examined in surveys of parental populations, which are less likely to have a bias due to ascertainment on the basis of abnormal phenotype. the frequencies in the postnatal and prenatal populations examined in this study were calculated to be 5.7 x 10(-5) (95% confidence interval is 3.2-8.2 x 10(-5)) and 4.1 x 10(-5) (95% confidence interval is 2.0-6.2 x 10(-5)). However, in view of the extent of variation reported in the various studies, these estimates should be considered first approximations of the true frequency.

We have investigated the capacity of a murine cell line with a temperature-sensitive (ts) mutation in the DNA polymerase alpha (Pola) locus and a series of ts non-Pola mutant cell lines from separate complementation groups to stimulate DNA synthesis, in senescent fibroblast nuclei in heterokaryons. In the Pola mutant x senescent heterodikaryons, both human and murine nuclei display significantly diminished levels of DNA synthesis at the restrictive temperature (39.5 degrees C) as determined by [3H]thymidine labeling in autoradiographs. In contrast, all of the non-Pola mutants, as well as the parental (wild type) murine cells, induced similar levels of DNA synthesis in both parental nuclei at the nonpermissive and permissive temperatures. Similarly, young human fibroblasts are also able to initiate DNA synthesis in heterokaryons with the ts Pola mutant at the two temperatures. In order to determine if complementation of the non-Pola mutants requires induction of serum responsive factors in the senescent cells, fusion studies of similar design were conducted with young and old human fibroblasts incubated in low serum (0.2%) for 48 hr prior to and after cell fusion. Again, a diminished level of DNA synthesis was observed at 39.5 degrees C in the Pola mutant x senescent cell heterokaryons. In these low-serum studies, both parental nuclei in the Pola x young cell heterokaryons and the human nuclei in heterokaryons with one of the non-Pola mutants (FT107) also displayed diminished levels of DNA synthetic activity. All of the other mutants are able to support similar levels of synthetic activity at both temperatures in the presence of reduced serum. The nature of the mutation in three of the non-Pola lines has not been determined but, like the Pola mutant cells, are inhibited in the G1 phase of the cell cycle when incubated at the nonpermissive temperature (39.5 degrees C). The fourth non-Pola mutant line is known to have at least one ts mutation in the cdc2 gene and is inhibited in the G2 phase when exposed to 39.5 degrees C. These results suggest that there may be a functional deficiency of pol alpha in senescent human fibroblasts, and this replication factor may be one of the rate-limiting factors involved in loss of the capacity to initiate DNA synthesis in senescent cells.

Findings in a patient with mosaic isochromosome 8p are compared with those of previously reported cases. There were no distinguishing findings on physical examination; all had cognitive delays, especially in speech and language development.

A simple and reproducible method for cryopreservation of brain tissue from patients with Alzheimer's disease is described. Fresh brain slices (1 cm thick) obtained less than 6 h postmortem are placed in sealed plastic bags, sandwiched between 0.3-cm-thick aluminium sheets, and frozen by placing the entire "sandwich" between layers of dry ice pellets. The frozen brain slices are stored at -85 degrees C. Specific anatomic areas can be retrieved at any time for light and electron microscopic, immunocytochemical, autoradiographic and neurochemical studies.

The limited proliferative potential of the cultured human diploid fibroblast is now well established. A number of biological correlates suggest that this culture system is a model for the study of aging at the cellular level. The mechanism(s) that causes the loss of proliferative activity is unknown; the results of some recent studies indicate that specific genes may play a pivotal role in cellular aging in vitro. The extent to which changes in proliferative functions are causally related to aging in vivo is currently under investigation.

We have previously reported that the DNA polymerase alpha activity/unit cellular protein is decreased in late-passage (senescent) human diploid fibroblast-like (HDFL) cultures due to the cellular enlargement associated with in vitro aging. In the studies described here, we have used cell fusion technology to investigate the formal kinetic relationship between the concentration of DNA polymerase alpha and the rate of reinitiation of DNA synthesis in nuclei from senescent cells. Heterokaryons were derived from the fusion of senescent cells to a series of actively dividing cell types with inherently different DNA polymerase alpha activities per cell. A kinetic analysis revealed a first-order relationship between the entry into S phase of senescent nuclei and the concentration of DNA polymerase alpha activity calculated to be in heterokaryons. This result suggests that increases in cell volume may be related to the decline in proliferative activity of late-passage HDFL cells, via "dilution" of factors essential for cellular replication.

We have examined the kinetic relationship between the rate of entry into the S phase in human diploid fibroblast-like (HDFL) monokaryon cells and (1) the concentration of DNA polymerase alpha activity and (2) the cell volume. In the former studies, a first-order dependence between the rate of entry into the S phase and the concentration of DNA polymerase alpha activity was observed, consistent with the enzyme, or a coregulated factor, being rate limiting for this metabolic process. Examination of the nature of the dependence of the rate of entry into the S phase upon cell volume revealed a more complex relationship. The results obtained in studies with synchronized cultures are consistent with the presence of two to three rate-limiting reactants when cell volume is the independent variable. Studies with asynchronous HDFL cell cultures revealed that the smallest cells in the G1 population, presumably the early G1 cells, enter the S phase at an increasing rate as a function of cell volume up to a certain size, beyond which the cells enter at a decreasing rate similar to that observed in the studies with the synchronized cultures. Similar studies examining the relationship between cell volume and the rate of entry into S phase in three established immortal cell lines revealed positive correlation between the rate of entry into S phase and cell volume throughout the size range of the G1 population. This latter observation suggests that the factors involved in the initiation of the S phase may be present in concentrations that are not rate limiting in immortal cell lines.

One variant, aphhs-3 was previously isolated based on a hypersensitivity to nontoxic concentrations of aphidicolin, a specific inhibitor of DNA polymerases-alpha and delta. This variant was found to be more sensitive to temperatures above 35 degrees C and to 10 microM of 3'-azido-3'-deoxythymidine (zidovudine, azidothymidine, or AZT) than the parental 743x cells. DNA polymerase activities in the cell extract or in the partially purified fraction by DEAE-cellulose (DE52) anion exchange column from aphhs-3 were active at 40 degrees C. No significant differences in deoxynucleoside triphosphate pools were observed at 34 degrees C for both the parental 743x and aphhs-3 cells. Revertants were isolated at 39 degrees C: six revertants (aphhs-3-tr1 through aphhs-3-tr6) were obtained without aphidicolin; one revertant aphhs-3-tar (the tar clone) was selected in aphidicolin (0.12 microM). The hypersensitivity to aphidicolin (Aphhs) and AZT (AZThs) was cosegregated in the revertant aphhs-3-tr5 (the tr5 clone), while the tar clone was not AZThs. There was a similar increase in the specific activity of 3H-labeled DNA in all cell lines after additions of [3H]AZT or [3H]thymidine. Additions of purine or pyrimidine arabinosides (araT, araC, and araA) to all cell lines resulted in a similar cytotoxicity, suggesting the anabolism of dTTP was not defective in the tr5 clone. The spontaneous mutation rate at the hypoxanthine-guanine phosphoryltransferase locus using replating techniques and 6-thioguanine resistance selection was less than or equal to 5 x 10(-7), 2.2 x 10(-6), or 1.3 x 10(-6) per generation for the tr5, 743x, or tar cell lines, respectively. Most importantly, DNA polymerase activities in the cell extract of the revertant tr5 clone were inhibited by 0.5 microM AZTTP. In contrast, no inhibition was observed in those of the parental 743x and revertant tar cells. The cosegregation of both Aphhs and AZThs in the tr5 revertant suggests that these two phenotypes may be a result of the same mutational event.

In vitro exposures of mass cultures and clones of human diploid fibroblastlike cells to erythromycin, in concentrations of 50 to 400 micrograms/ml, results in increasing degrees of growth inhibition and augmented cell volume, with a shift toward larger proportions of cells of the epithelioid type and fewer of the fibroblast type. These alterations were reversed upon subculture in the absence of the antibiotic.

The last decade has witnessed increasing application of human cytogenetic technology to prenatal chromosome analysis. However, unlike the rather uniform peripheral blood T-lymphocyte system which has provided most of our experience in human cytogenetics, long-term amniotic-fluid cell cultures display extreme cellular heterogeneity and disproportionate growth of certain cell types as a consequence of clonal amplification. When they enter cell culture, many of these cells are approaching the terminal stages of their respective, life spans and may have accumulated chromosomal aberrations. Concern about the possibility of true fetal mosaicism seems warranted chiefly in situations were multiple colonies display potentially viable aberrations. Clonal analysis, preferably of multiple clonal types, and attention to details of clonal morphology are likely to minimize diagnostic errors and undue apprehension resulting from mosaicism in amniotic-fluid cell cultures.

Barrett's esophagus is a condition in which metaplastic columnar epithelium replaces squamous esophageal epithelium as a consequence of chronic gastroesophageal reflux. Patients with this condition are at increased risk for the development of adenocarcinoma. To better understand the progression to adenocarcinoma in this disease, we studied abnormalities in DNA content of epithelial cells in Barrett's esophagus. Using flow cytometry, we examined the spatial distribution of abnormal nuclear DNA contents (aneuploidy) in the esophagi of 14 patients with Barrett's adenocarcinoma. Multiple (2 to 14) populations of aneuploid cells were seen in 12 of the 14 cases. Some early carcinomas appeared to be associated with a single aneuploid population of cells. Surrounding dysplastic epithelium often contained multiple, different overlapping aneuploid populations. These data suggest that neoplastic progression in Barret's esophagus is associated with a process of genomic instability which leads to evolution of multiple aneuploid populations, with the ultimate development of a clone of cells capable of malignant invasion. Thus, detection of multiple aneuploid populations of cells in Barrett's esophagus may indicate a high risk of cancer. Barrett's esophagus provides a unique and readily accessible model for the study of neoplastic progression in human epithelial malignancy.

Werner's syndrome is commonly regarded as a model for the study of premature aging. There are, however, a variety of clinical and pathologic anatomical features that clearly distinguish it from aging in normal individuals. In this paper we report on in vitro cytogenetic and cell fusion studies that indicate cultured fibroblast-like cells derived from Werner patients differ from cells of normal donors. Despite these discordances with "natural" aging, however, Werner's syndrome, like several other "segmental progeroid syndromes," may prove useful for the investigation of selected aspects of the aging process and of age-related diseases.

It has previously been shown that serum-deprived, early passage quiescent human diploid fibroblastlike (HDFL) cells are able to inhibit cycling cells from entry into DNA synthesis upon cell fusion. We have found that the degree of inhibition of DNA synthesis in the heterokaryon correlates with the duration of serum deprivation, which is consistent with the suggestion that serum-deprived cells may enter progressively deeper stages of G0 as they increase their time in quiescence. In contrast to fusions with senescent cells, in heterokaryons between serum-deprived early passage and cycling young cells transient inhibition of protein synthesis with cycloheximide or inhibition of RNA synthesis with 5-6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) did not stimulate nuclear [3H]-thymidine incorporation. These results suggest that differences may exist in the mechanisms responsible for inhibiting cell cycle progression in senescent vs early passage quiescent HDFL cells.

We have isolated a diploid fibroblast culture from human fetal lung with an in vitro lifespan of about 100 population doublings. The culture grows very well at clonal densities and long-lived clones can be isolated for use in cellular aging studies. The longer in vitro lifespan of the culture has allowed us to isolate from it a clone, containing a dominant and recessive mutation, having significant remaining proliferative potential. The nature of the mutations will allow for hybrid selection, after fusion of the mutant clone with wild type human cells. The mass culture and clones derived from it provide a valuable resource for cell aging studies.

The term "variegated translocation mosaicism" is used to describe the repeated occurrence, within cultures of human skin fibroblasts, of a multiplicity of chromosomal rearrangements. With respect to the frequencies of such cytogenetically aberrant clones we found that they (1) were not detectable in routine diagnostic skin fibroblast cultures from 29 subjects with a wide variety of indications for biopsy; (2) were not detectable during in vitro aging of diploid strains with four normal individuals; (3) could be detected after rescue from bacterial contamination of a culture from an otherwise normal diploid male; (4) occurred with high frequencies in independent cultures from another apparently normal subject; (5) occurred with high frequencies in multiple biopsies obtained at autopsy from a patient with Werner's syndrome who died of sepsis; (6) were of pseudodiploid nature; and (7) involved a different spectrum of chromosomes in different individuals. A consistent association with mycoplasma contamination could not be made.

Observations of the growth kinetics and morphologies of clones and subclones of diploid human skin fibroblast cultures lead to the working hypothesis that these cells, presumably like their counterparts in healing wounds, constitute a differentiating system. There is attenuation of the growth of serial clones, with continual selection for more vigorous stem cells. The latter segregate daughter cells of varying growth potential, including a class of cells which may be regarded as terminally differentiating; we propose that such cells may be histiocytes or macrophages. These studies a) demonstrate extensive epigenetic heterogeneity in fibroblast cultures, b) suggest that hyperplastic foci may be monoclonal or oligoclonal, c) rule out a simple biologic clock mechanism as an explanation of clonal senescence, d) suggest a new approach to the analysis of various inborn errors of metabolism, such as Werner's Syndrome.

Chicken myogenic stem cells can undergo symmetric and asymmetric cell divisions. Symmetric divisions produce two stem cells or two cells committed to terminal muscle differentiation. Asymmetric divisions produce one stem cell and one committed cell. Committed cells undergo four divisions, and their progeny differentiate into postmitotic, biochemically distinct muscle cells, which can be identified immunocytochemically. The control of stem cell commitment was investigated in vitro by means of cell cloning and subcloning experiments, and computer modeling. We found that stem cell commitment is a process which can be modeled as a stochastic event, with a central tendency or probability of 0.2 +/- 0.1. This value is independent of organismal or mitotic age of the stem cells, cell density, or growth in a mitogen-poor environment. Myogenic stem cells stop dividing after approximately 30 divisions in vitro. Since the probability of commitment to terminal differentiation remains below 0.5, clonal senescence and terminal differentiation are separate processes in this system.

The growth of primary clones and serial subclones of HeLa cells and of diploid human fibroblast-like cells were compared both in the presence and absence of feeder layers; the latter had no significant effects upon the results. Clones and subclones of both cell types displayed great heterogeneity in growth rates, typically with a bimodality of growth distributions. Serial passages of clones selected on the basis of superior rates of proliferation showed attentuation of growth potentials; the extent of such attentuations was much less in the case of HeLa cells, suggesting at least one possible basis for the differences in long-term growth potential between these two classes of cell lines.

We previously demonstrated an inverse relationship between the G1 volume of human diploid fibroblast-like (HDFL) cells obtained from foreskin tissue and clonal replicative potential. On the basis of these results, we suggested that one process underlying in vitro senescence is a progressive increase in the mean cell volume of successive progeny within clonal lineages. We now report that the size of HDFL cells, as well as of chick embryo fibroblasts, can be increased in the virtual absence of cell division by culturing at low density and at low serum concentration (0.1-1.0%). Consequent to an increase in cell size, the replicative potential of the cells is reduced to the level of later-passage cells of similar size. By clonal analysis, the populations of enlarged cells contain up to three times as many nondividing cells as do controls. In the enlarged populations, the proportion of cells producing attenuated clones (four or fewer progeny) increases by about 30%, whereas the proportion of cells yielding greater than 32 cells declines by a similar percentage. These observations lead us to propose that replicative potential may be limited by cell size, which in turn may be regulated by a kinetic relationship between cellular growth and cell division cycles.

Four patients with features suggestive of chromosome disorders but with normal lymphocyte karyotypes were found to have chromosome aberrations in skin fibroblast karyotypes. Although mosaicism for chromosome abnormalities in lymphocyte cultures is common, apparent restriction of mosaicism to one tissue is unusual. We suggest that after examination of lymphocyte karyotypes, certain patients warrant cytogenetic evaluation of a second tissue, usually cultured skin fibroblasts.

Previous studies have demonstrated that upon fusion with actively cycling human diploid fibroblast-like (HDFL) cells, senescent HDFL are capable of inhibiting the young nucleus from entry into DNA synthesis (Norwood et al. (1974) [10]; Rabinovitch et al. (1980) [1]). These studies have been interpreted as evidence in favor of the existence of cell cycle inhibitors in senescent cells. Enucleate cytoplasts derived from senescent HDFL cells are also capable of inhibiting the young nucleus from entry into DNA synthesis upon fusion with whole cycling HDFL cells. In contrast, no inhibition was observed in fusions between enucleate young cells and whole cycling cells. These results provide evidence for transmissible cell cycle-inhibitory factors in the cytoplasm of senescent cells.

In heterokaryons between senescent and young diploid fibroblast-like cells, dominance of the former with respect to nuclear DNA synthesis (incorporation of [(3)H]thymidine) was demonstrated. For identification of the respective partners, double-layer autoradiography was used after the old cells were labeled with [(3)H]methionine and the young cells were labeled with [(14)C]thymidine. Synchrony of nuclear labeling (i.e., all nuclei in a cell labeled with [(3)H]thymidine) was observed in the majority of di- and polykaryons during the second and third of three 24-hr periods of labeling with [(3)H]thymidine. The results are compatible with either terminal differentiation or error theories of clonal senescence.

Trisomy for an F-like chromosome with banding characteristics of chromosome 20 was observed, respectively, in 25 and 8% of amniotic fluid cell clones in two cases of prenatal diagnosis for advanced maternal age. No such aneuploidy was found upon examination of fetal or postnatal tissues. In both instances there were no detectable malformation. It is suggested that propensity to nondisjunction for an F-like chromosome, not unlike tetraploidy, might be another characteristic of amniotic fluid cells in culture.