A p53 dose-response relationship for sensitivity to DNA damage in isogenic teratocarcinoma cells.

Teratocarcinomas are tumors that arise from primordial germ cells and are readily curable with DNA-damaging chemotherapeutic drugs. Teratocarcinoma cells ex vivo in tissue culture are also relatively chemosensitive and undergo apoptotic death in response to DNA damage. We have previously hypothesized that the observed sensitivity of this tumor type to DNA damage is related to high basal expression of wild-type p53 protein. We have now addressed this issue by characterizing the DNA damage response of isogenic teratocarcinoma cells that differ only in their level of expression of wild-type p53 protein. We find a clear p53 dose-response relationship in these cells for rapid apoptosis following DNA damage that correlates with diminished colony formation in clonogenic survival assays. These results suggest that strategies to increase basal wild-type p53 protein expression prior to treatment with DNA-damaging drugs may improve curability in other tumor types.

Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay.

Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer. Numerous studies indicate that 'hot-spot' p53 mutants (which comprise approximately 30% of human p53 gene mutations) are largely devoid of transcriptional activity. However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites. We have modified a previously described yeast-based p53 functional assay to readily identify such partial loss of function p53 mutants. We demonstrate the utility of this modified p53 functional assay using a diverse panel of p53 mutants.

Testicular germ cell tumors: molecular understanding and clinical implications.

It was recognized in the 1960s that testicular germ cell tumors were curable with chemotherapeutic drugs. Since that time, newer drugs including cisplatin have increased the cure rate of these tumors to over 80%, even in patients with metastatic disease. Germ cell tumors also exhibit a unique biology and genetics that distinguish them from other solid tumors and might contribute to their routine curability.

P53 tumour suppressor gene and germ cell neoplasia.

P53 tumour suppressor gene mutations occur in approximately 50% of common solid tumours such as breast, colon and lung. In contrast, p53 gene mutations occur infrequently (< 3%) in germ cell tumours, even though p53 protein is expressed at high levels in the vast majority of tumour samples. P53-regulated genes are not correspondingly over-expressed in germ cell tumour samples and cell lines, indicating that p53 functions poorly as a transcription factor in this tumour type. High levels of wild-type p53 may contribute, in part, to the chemosensitivity of germ cell tumours.

Peripheral blood progenitor cell cycle kinetics following priming with pIXY321 in patients treated with the "ICE" regimen.

PURPOSE: Treatment with hematopoietic growth factors increases the percentage of hematopoietic progenitor cells in cell cycle. Following withdrawal of certain growth factors, preclinical data suggest that there is a transient fall in the percentage of progenitor cells in cycle below the baseline, thus providing a window to administer chemotherapy with reduced risk of myelotoxicity. PATIENTS AND METHODS: Patients with histologically confirmed, previously untreated neoplasia, were treated with pIXY321 by subcutaneous injection at a dose of 375 microg/m2 twice daily (total dose 750 microg/m2/day) for seven days (days -8 to -2), followed by a two-day rest (days -1 to 0). Patients received ICE (ifosfamide, carboplatin and etoposide) on days 1 to 3. On day 4, pIXY321 was resumed until hematologic recovery. Peripheral blood was collected on days -8, -2, -1, 1, and cell cycle distribution was determined using flow cytometry. RESULTS: Twenty patients were treated in this study and received a total of 54 cycles. Partial responses were observed in three of 13 patients with non-small cell lung cancer (23 percent) and two of five patients with small cell lung cancer (40 percent). Six of 15 patients had an increased number of cells in S+G2/M on day 1 of ICE following seven days of pIXY321 and two days off (days -1 to 0). The average increase was 63 percent (range 6-253). Seven patients had a decreased number of cells in S+G2/M. The average decrease was 55 percent (range 6.3-78). There were no significant differences among the fifteen patients with regards to the observed toxicity of the chemotherapy. DISCUSSION: pIXY321 in this schedule did not consistently decrease the percentage of cycling progenitor cells in the peripheral blood. Future studies should define whether other growth factors and/or schedules can synchronize progenitor cell cycling and protect the marrow compartment from cycle specific chemotherapy.

A functionally inactive p53 protein in teratocarcinoma cells is activated by either DNA damage or cellular differentiation.

Testicular teratocarcinomas never contain p53 gene mutations even though these tumors express high levels of nuclear p53 protein. We have characterized two murine teratocarcinoma cell lines and find no evidence that endogenous p53-regulated genes are correspondingly upregulated. Differentiation of these teratocarcinoma cells with retinoic acid results in a marked decrease in p53 protein levels but is accompanied by a marked increase in p53-mediated transcriptional activity. Together these results support the hypothesis that the p53 protein in undifferentiated teratocarcinoma cells is transcriptionally inactive and accounts for the lack of selection for p53 gene mutations in this tumor type. These teratocarcinoma cells undergo p53-mediated apoptosis in response to DNA damage, which may explain the routine cures of human testicular tumors with combination chemotherapy.

Structure and expression of germ line immunoglobulin heavy-chain epsilon transcripts: interleukin-4 plus lipopolysaccharide-directed switching to C epsilon.

We have isolated cDNA clones complementary to a truncated immunoglobulin heavy-chain C epsilon RNA transcript previously found to be induced in B lymphoid cells by treatment with lipopolysaccharide (LPS) combined with interleukin-4 (IL-4). We demonstrate that this transcript initiates from a promoter upstream of the germ line epsilon class-switch recombination region (S epsilon region). The major germ line C epsilon transcript contains a small 5' exon contributed by sequences upstream of the S epsilon region spliced to the normal C epsilon exons. Treatment of splenic B lymphoid cells with LPS plus IL-4 induces the expression of transcripts from the germ line epsilon transcription unit followed by expression of normal immunoglobulin epsilon heavy-chain mRNA. Furthermore, we demonstrate that similar treatment of transformed precursor B cell lines induces the expression of germ line epsilon transcripts followed by class switching to epsilon expression in these lines. This is the first demonstration of switching to epsilon in cells of the pre-B stage. The general structure of the germ line epsilon transcript and transcription unit is similar to that previously characterized for germ line gamma 2b transcripts. However, expression of these two germ line transcription units in B-lineage cells is inversely regulated by IL-4 (plus LPS) treatment, correlating with the effects of these treatments on switching to these loci.

Structure and expression of germline immunoglobulin gamma 3 heavy chain gene transcripts: implications for mitogen and lymphokine directed class-switching.

We have characterized the structure and expression of transcripts synthesized from the murine germline immunoglobulin gamma 3 heavy chain gene in certain B-lineage cells. The transcripts initiate upstream of the switch gamma 3 region, generating a 5' exon that is spliced to C gamma 3 exons. Expression of this germline transcript is induced when splenic B cells or A-MuLV-transformed pre-B cell lines are cultured in the presence of lipopolysaccharide (LPS). Addition of interleukin-4 (IL-4) to these lipopolysaccharide (LPS) cultures dramatically inhibits induction of the germline gamma 3 transcript. Induction of germline gamma 3 transcripts occurs before the increased accumulation of gamma 3-producing cells and VDJ-gamma 3 mRNA in cultures of splenic B cells. These data provide further evidence that germline CH transcriptional units are important components in the regulation of heavy chain class-switching. In addition, the pre-B cell lines that we describe represent the first example of permanent cell lines that regulate expression of the germline gamma locus in response to LPS plus IL-4 treatment in a manner analogous to normal B cells; therefore these lines should represent an excellent model system to further study the molecular mechanisms by which germline expression is regulated by these agents.

Mitogen plus interleukin 4 induction of C epsilon transcripts in B lymphoid cells.

To elucidate the mechanism of IL-4-induced enhancement of IgE and IgG1 production, murine splenic B cells and A-MuLV-transformed cells were cultured with LPS and IL-4 and assayed for epsilon and gamma 1 transcripts. Concomitant treatment with IL-4 and LPS induced expression of C epsilon transcripts in both normal and transformed cells. Expression of these truncated C epsilon transcripts preceded accumulation of normal epsilon mRNA in treated cells. Consistent data were obtained with respect to gamma 1 RNA expression. These results suggest that IL-4 can direct class switching in the context of a mechanism associated with differential expression of germline constant region genes.

Mitogen- and IL-4-regulated expression of germ-line Ig gamma 2b transcripts: evidence for directed heavy chain class switching.

Treatment of murine B cells with bacterial lipopolysaccharide (LPS) in the presence or absence of different lymphokines results in cell populations that differentially express particular immunoglobulin heavy chain constant region (CH) genes. This class switch involves recombination between switch regions located upstream of the germ-line CH genes. We have treated Abelson murine leukemia virus-transformed pre-B cells and normal splenic B cells with LPS or LPS plus the lymphokine IL-4 and examined the effect on the germ-line gamma 2b locus and gamma 2b class switching. In both cell types, LPS induces transcription specifically through the germ-line gamma 2b locus before gamma 2b class switching. Furthermore, IL-4 inhibits LPS induction of germ-line gamma 2b transcripts in spleen cells and correspondingly abrogates switching to this CH gene. Thus treatment with mitogens and lymphokines can alter transcription of germ-line CH genes in B lineage cells and thereby directly regulate class switching in the context of a recombinase accessibility mechanism.

Structure and expression of germ line immunoglobulin gamma 2b transcripts.

We have isolated a cDNA copy of a truncated C gamma 2b transcript produced by Abelson murine leukemia virus transformants that spontaneously switch from mu to gamma 2b. The initiation site of this transcript was 2 kilobases 5' to the gamma 2b switch recombination region, demonstrating its germ line origin. Nucleotide sequence analyses suggest that this transcript does not encode a protein. Expression of germ line gamma 2b transcripts in Abelson murine leukemia virus transformants and in normal spleen cells correlated with endogenous gamma 2b class switch activity.

Control of recombination events during lymphocyte differentiation. Heavy chain variable region gene assembly and heavy chain class switching.

Our recent studies have focused on the organization of immunoglobulin genes in mice and humans and the mechanism and control of the recombination events that are involved in their assembly and expression. This report describes our progress in this area with particular focus on elucidating factors that influence the generation of the antibody repertoire in normal and diseased states. We present a detailed analysis of the organization of the human VH locus, studies that help to elucidate the nature of the recombination defect in mice with severe combined immunodeficiency, and studies of transgenic mice that focus on the mechanism that regulates tissue-specific variable region gene assembly. In addition, we also characterize mechanisms that control the heavy chain class-switch process. Although the latter process apparently involve a recombination system distinct from that involved in variable region assembly, we find that the two recombination events appear to be controlled by similar mechanisms.

Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching.

We describe rearrangement events which alter expression from a productive VHDJH rearrangement in an Abelson murine leukemia virus-transformed pre-B cell line. One such rearrangement results in replacement of the initially expressed variable region gene by a site-specific join between the open reading frame of a LINE-1 repetitive element and a remaining JH segment. We discuss this event in the context of the 'accessibility' model of recombinase control, and with respect to similar rearrangements involved in oncogene activation. In another subclone of the same pre-B cell line, altered heavy chain expression resulted from a mu to gamma 2b class switch recombination which occurred by a recombination-deletion mechanism but involved a complex inversion. We provide evidence that the germline gamma 2b region is specifically expressed in pre-B cell lines and early in normal development. We propose that the predisposition of pre-B cell lines to switch to gamma 2b production may reflect a normal physiological phenomenon in which the switch event is directed by an increased 'accessibility' of the germline gamma 2b locus to switch-recombination enzymatic machinery. Our findings support the hypothesis that the apparently distinct recombination systems involved in variable region gene assembly and heavy chain class switching are both directed by the accessibility of their substrate gene segments.

Recombination between immunoglobulin variable region gene segments is enhanced by transcription.

Immunoglobulin (Ig) variable (V) region genes are assembled in precursor B (pre-B) lymphocytes from multiple germline segments. The heavy-chain V-region gene is composed of variable (VH), diversity (D) and joining (JH) segments; kappa (K) and lambda (lambda) light-chain V-region genes have analogous VL and JL segments. Assembly of Ig V-gene segments, as well as those of the highly related T-cell receptor, is regulated at several levels and shows both stage and tissue specificity; for example Ig heavy-chain V-gene assembly precedes that of Ig light chains during B-cell differentiation. Joining of all classes of V-gene segments involves conserved recognition sequences that are probably targets for a common recombinase. Evidence has been presented suggesting that rearrangement of specific classes of segments is regulated by modulation of their accessibility to the recombinase. To elucidate mechanisms which control V-region gene assembly, we have investigated the effect of flanking gene expression on the frequency at which introduced V-gene segments are assembled in pre-B cell lines. Our findings suggest that transcription may play a direct role in the regulation of immunoglobulin V-gene assembly.

Molecular basis of heavy-chain class switching and switch region deletion in an Abelson virus-transformed cell line.

We demonstrated that a subclone of an Abelson murine leukemia virus-transformed B-lymphoid cell line switched from mu to gamma 2b expression in vitro, by the classical recombination-deletion mechanism. In this line, the expressed VHDJH region and the C gamma 2b constant region gene were juxtaposed by a recombination event which linked the highly repetitive portions of the S mu and S gama 2b regions and resulted in the loss of the C mu gene from the intervening region. An additional recombination event in this subclone involved an internal deletion in the S mu region of the expressed (switched) allele. One end of this deletion occurred very close to the switch recombination point. Despite the recombination-deletion mechanism of switching, the gamma 2b-producing line retained two copies of the C mu gene and two copies of the sequence just 5' to the S gamma 2b recombination point. The possible significance of the retention of these sequences to the mechanism of class switching is discussed.

Evaluating strategies to improve careprovider performance on health and developmental tasks in an infant care facility.

Responding to administrative staff and parental concerns, using modified reversal and withdrawal designs, two experiments evaluated a staff-managed feedback system to improve the hygiene and developmental skills of children in an infant/toddler center. Experiment 1 examined feedback designed to increase staff performance in checking and changing diapers, and recording those changes. A chart plus supervisory feedback produced increases in and maintenance of staff performance. Experiment 2 compared an existing staff management system with a "playchart" plus feedback in increasing careprovider-infant stimulation. The data (with follow-up on a new staff) supported the use of the new feedback system. Questionnaire data further supported the utility of the playchart system.

Radiological signs of acute pancreatitis.

Acute pancreatitis is commonly presented as a confusing and challenging diagnostic problem. This article is intended to show the increasingly important contribution radiology plays in the establishment of a correct initial diagnosis as well as in recognizing the serious complications of acute pancreatitis. A short discussion of the etiology and pathophysiology of pancreatitis is essential in understanding the mechanism of production of the X-ray findings. The most important recently published papers on this topic are reviewed together with a personal evaluation of the most significant and reliable radiographic changes. Statistical data, indications, and contraindications as well as the limitations of our radiographic procedures are discussed.