Recognizing factitious hypoglycemia in the family practice setting.

BACKGROUND: Factitious hypoglycemia is a deliberate attempt to induce a low serum glucose level using either insulin or oral hypoglycemic agents. Sulfonylurea-induced hypoglycemia is more common than incidents of insulin abuse, and hypoglycemia caused by these oral agents is biochemically indistinguishable from insulinoma. METHODS: We describe a case of factitious hypoglycemia resulting from insulin abuse in an adult diabetic patient, review the essentials of glucose homeostasis, and describe diagnostic tests that allow a differential diagnosis. RESULTS AND CONCLUSION: Factitious hypoglycemia is associated with a higher incidence of suicide, depression, and personality disorders. Insulin-induced hypoglycemia can be detected by an insulin to C-peptide ratio that is greater than 1.0. In the absence of proof to the contrary, insulinoma should be considered the cause of hypoglycemia until another diagnosis is established. The generally poor prognosis for patients with factitious hypoglycemia underscores the importance of early recognition of factitious disorders.

Difficult cases in heart failure: Reversible heart failure secondary to thyrotoxicosis.

Perhaps one of the most important steps in caring for a patient with heart failure is seeking a reversible form of heart failure. Often addressing a disease process, like myocardial ischemia or systemic hypertension, rapidly attenuates the progressive natural history of left ventricular dysfunction. Beyond the usual reversible causes, however, are some that may be overlooked. This case provides an insight into one of these causes. (c)1999 by CHF, Inc.

RREB-1, a novel zinc finger protein, is involved in the differentiation response to Ras in human medullary thyroid carcinomas.

An activated ras oncogene induces a program of differentiation in the human medullary thyroid cancer cell line TT. This differentiation process is accompanied by a marked increase in the transcription of the human calcitonin (CT) gene. We have localized a unique Ras-responsive transcriptional element (RRE) in the CT gene promoter. DNase I protection indicates two domains of protein-DNA interaction, and each domain separately can confer Ras-mediated transcriptional inducibility. This bipartite RRE was also found to be Raf responsive. By affinity screening, we have cloned a cDNA coding for a zinc finger transcription factor (RREB-1) that binds to the distal RRE. The consensus binding site for this factor is CCCCAAACCACCCC. RREB-1 is expressed ubiquitously in human tissues outside the adult brain. Overexpression of RREB-1 protein in TT cells confers the ability to mediate increased transactivation of the CT gene promoter-reporter construct during Ras- or Raf-induced differentiation. These data suggest that RREB-1 may play a role in Ras and Raf signal transduction in medullary thyroid cancer and other cells.

Expression of prokaryotic HhaI DNA methyltransferase is transforming and lethal to NIH 3T3 cells.

In neoplastic cells, levels of DNA methyltransferase activity are often increased, and evidence is accruing to suggest an important role for this event in tumorigenesis. To evaluate this possibility further, and to investigate the contribution of increasing de novo, as opposed to maintenance, DNA methylation in mammalian cells, we expressed the bacterial HhaI methyltransferase in cultured murine fibroblasts. This enzyme is a pure de novo DNA methyltransferase that methylates the internal C in the sequence GCGC. We find that both constitutive and induced expression of the wild-type HhaI results, primarily, in lethality to the cells. However, surviving cell clones that express low levels of M. HhaI demonstrate increased tumorigenicity as assessed by soft agar cloning efficiency (8.6% for sense HhaI-transduced PA 317 cells versus 0.4% for antisense controls; 1.7% for sense HhaI-transfected NIH 3T3 cells versus 0% for a mutant HhaI control) and tumorigenicity in nude mouse heterotransplants (75% for sense HhaI-transduced PA 317 cells versus 18.5% for antisense controls). DNA isolated from the clonogenic sense HhaI clones, versus clones expressing the mutant HhaI gene, has no increase in overall CpG methylation but an average of 27% (range, 16.7-38.9) increase in methylcytosine content at GCGC sites. These findings suggest that eukaryotic cells tolerate a narrow window of increase de novo DNA methylating capacity, above which cell death occurs and within cell transformation results. Our results further emphasize the potential role of increased DNA methyltransferase activity in the evolution of cancer.

Treatment of advanced medullary thyroid carcinoma with a combination of cyclophosphamide, vincristine, and dacarbazine.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a neoplasm of the parafollicular C cells of the thyroid gland, which belongs to the diffuse neuroendocrine system. This cancer usually behaves in a relatively indolent manner for most patients. However, approximately 20% of patients have a more aggressive course that requires effective management. There are few reported clinical trials of chemotherapy for MTC. From the literature, the most active agent appears to be doxorubicin, with response rates of 30% reported. On the basis of the activity of cyclophosphamide, vincristine, and dacarbazine (CVD) in other advanced neuroendocrine neoplasms, the authors tested the combination in patients with advanced MTC. METHODS: Seven patients with advanced MTC were treated with cyclophosphamide (750 mg/m2), vincristine (1.4 mg/m2), and dacarbazine (600 mg/m2 daily for 2 days in each cycle) every 3 weeks. Assessments of measurable tumor and serum calcitonin and carcinoembryonic antigen were made before treatment and followed up until progressive disease was documented. RESULTS: Two patients had partial tumor and biochemical responses for a duration of 14 and 29 months, respectively. One patient had a partial biochemical response and stable tumor measurements for 9 months, and another patient had stable tumor size and markers for 14 months. Three patients had progressive disease. Diarrhea and flushing improved in two patients who had partial biochemical responses. CONCLUSION: Our experience suggests that CVD chemotherapy has moderate activity and is well tolerated in patients with advanced MTC. Additional prospective studies of this regimen for MTC are required.

Pathobiology of the C-cells.

This brief review of the pathobiology of C-cells has stressed cellular and molecular aspects of MTC development. In the genetic forms of MTC, an alteration in one or more genes on chromosome 10, through an as yet unknown series of events, results in initial hyperproliferation of C-cells. Subsequent genetic steps, possibly at other chromosome loci, presumably result in selected clonal transformation of the hyperproliferative C-cells at risk for tumor development. Some of these same molecular events are probably operative in the development of sporadic MTC as well. Once MTC has developed, it has the potential to undergo tumor progression events which result in loss of C-cell differentiation. Studies in a culture model of these events have revealed that activation of signal transduction pathways, similar to those active in differentiation of other neural crest-derived cells, can restore differentiation features of normal C-cells to MTC. Continued identification of the molecular factors mediating this restoration should teach us much about the relationships between general neural crest differentiation and that of normal C-cells. It will also reveal much about the pathobiology of C-cells contributing to each step of MTC development.

Regulation of human calcitonin gene transcription by cyclic AMP.

Transcription of the human calcitonin (CT) gene is markedly increased by cAMP in the TT line of medullary thyroid carcinoma. This response is conferred by 5' flanking DNA sequences located between -132 and -252 relative to the transcription initiation site. Within this region are an upstream cyclic AMP response element (CRE), a downstream CRE flanked by two octamer motifs, and two adjacent C-rich AP2-like sequences. In transfection experiments in TT cells, the downstream CRE, combined with CT promoter sequences, generated 70% of the maximal cAMP response. The upstream CRE and the C-rich elements conferred 10 and 30% of this response, respectively. In gel mobility shift assays, specific TT cell proteins bound to each of these sequences. Therefore, the cAMP response of the CT gene is complex, requiring multiple elements acting in concert.

Human calcitonin gene regulation by helix-loop-helix recognition sequences.

Human calcitonin (CT) gene transcription is regulated by proximal 5' flanking sequences which mediate cAMP-induced expression, and by a distal basal enhancer region. Using transient expression of CT-CAT constructs, we showed that the basal enhancer is active in a CT-producing small cell lung cancer cell line (DMS53) and the thyroid C cell derived tumor line, TT, but is inactive in non-CT-producing cell lines. In deletional and direct mutational analyses of the distal enhancer region, disruption of two elements resembling recognition sequences for the helix-loop-helix (HLH) family of transcriptional regulatory proteins resulted in a significant loss of basal transcriptional enhancer action. These results suggest that HLH recognition motifs may mediate a significant portion of constitutive CT gene transcriptional activity in these cells. Nuclear protein extracts from DMS53 cells formed specific binding complexes with oligonucleotides containing two of these candidate enhancer sequences. However, proteins capable of binding to these CT gene HLH consensus recognition sites were not restricted to CT-producing cells. We conclude that members of the HLH protein family, some expressed ubiquitously and some expressed or activated in a tissue-restricted fashion, may combine to enhance CT gene transcription in tumor cells of neuroendocrine derivation.

Abnormal patterns of DNA methylation in human neoplasia: potential consequences for tumor progression.

An imbalance of DNA methylation, involving widespread hypomethylation, regional hypermethylation and increased cellular capacity for methylation, is characteristic of human neoplasia. This imbalance begins in preneoplastic cells and becomes more extensive throughout subsequent stages of tumor progression. In normal cells, a primary function of DNA methylation may be to modulate compartmentalization of DNA to ensure that regional areas of transcriptionally active chromatin replicate earlier than the bulk transcriptionally inactive chromatin. We argue here that the altered methylation patterns observed during tumor progression, especially regional hypermethylation, may mark--or even help to establish--abnormalities of chromatin organization. In turn, these changes in chromatin structure may, through direct transcriptional inactivation of genes, predisposition to mutations, and allelic deletions, mediate the progressive losses of gene expression associated with tumor development.

Differential utilization of calcitonin gene regulatory DNA sequences in cultured lines of medullary thyroid carcinoma and small-cell lung carcinoma.

Regulation of expression of the human calcitonin gene was found to differ between two tumor lines of different tissue origin, medullary thyroid carcinoma (TT line) and small-cell lung carcinoma (DMS53 line). Distal 5' DNA elements between -750 and -2000 exhibited a stronger basal activity in DMS53 than in TT cells, whereas proximal DNA sequences between -132 and -252 mediated a dramatic cyclic AMP response in TT but not DMS53 cells.

Changes in calcitonin gene RNA processing during growth of a human medullary thyroid carcinoma cell line.

The ratios of calcitonin (CT) to calcitonin gene-related peptide (CGRP) mRNA, both generated by alternative RNA processing from the same primary RNA transcript, are shown by Northern blotting of cytoplasmic RNA to vary as a function of growth in a human medullary thyroid carcinoma cell line (TT). Upon initial seeding, CT mRNA levels are relatively high, and CGRP mRNA levels are relatively low. During the early logarithmic growth phase, CGRP mRNA levels rise severalfold, while CT mRNA levels change only slightly. As the cells approach confluence, both CT and CGRP mRNA levels rise. Subsequently, CGRP mRNA levels fall substantially in postconfluent cells, while CT mRNA levels remain high. By actinomycin D blocking of nascent transcription, we have shown that these growth-related, reversible changes in the ratio of CT to CGRP mRNA are not due to changes in mRNA stability. Our data rather suggest that TT cells reversibly alter alternative RNA-processing patterns dependent upon growth conditions in vitro, such that CT mRNA is lowest and CGRP mRNA is highest during rapid growth. The mechanisms underlying this RNA-processing alteration may play a role in certain patients with aggressive forms of medullary thyroid carcinoma, in whom a decrease or loss of CT levels heralds a poor prognosis.

Low incidence of loss of chromosome 10 in sporadic and hereditary human medullary thyroid carcinoma.

Genetic linkage has been recently documented between a centromeric region of chromosome 10 and familial multiple endocrine neoplasia type II (MEN II). This syndrome consists of initial thyroid C-cell and adrenal chromaffin cell hyperplasia which result in multifocal medullary thyroid carcinomas and bilateral adrenal pheochromocytomas. Other hereditary cancers, such as retinoblastoma, appear to result from a series of genetic events involving, first the inheritance of a germ line abnormality, and subsequent loss of chromosome loci opposite this initial defect. In these cancers, this loss of the normal alleles in both familial and sporadic cases, is frequently manifest as a reduction to homozygosity for polymorphic DNA markers near the involved locus. It might then be expected that chromosome 10 regions would be lost with high frequency in tumor DNA from patients with MEN II and sporadic medullary thyroid carcinoma (MTC). We now demonstrate that only two of 16 MTC tumors studied by analysis of restriction fragment length polymorphisms for multiple regions of the short and long arms of chromosome 10 showed loci reduced to homozygosity. One of these tumors was from a patient with MEN II and the other from a patient with nonfamilial MTC. Importantly, no such chromosome 10 changes were noted in pheochromocytomas from the patient with MEN II or his sister. These findings strongly suggest that the sequence of genetic events for familial MTC is either different from that for retinoblastoma or that loss of normal alleles opposite the germ line genetic defect occurs by mechanisms other than gross loss of chromosomal material in MTC. A model is proposed suggesting that the mechanism involving loss of alleles opposite one another is operative in hereditary tumors, such as retinoblastoma, which do not arise within a setting of initial polyclonal cellular hyperplasia. In contrast, in tumors such as familial MTC and polyposis coli which arise as individual clones of neoplastic cells from a setting of preexistent polyclonal hyperplasia, the first genetic event may underlie hyperplasia, and additional events, frequently at other chromosomal loci, may cause individual clonal neoplasms.

The molecular biology of medullary thyroid carcinoma. A model for cancer development and progression.

Medullary thyroid carcinoma (MTC) is an important human cancer for the study of molecular abnormalities that underlie initiation of neoplasia and subsequent cellular changes during tumor progression. This tumor can occur in different inherited forms, each mediated by autosomal dominant genetic events. Germline abnormalities on chromosome 10 are linked to at least one type of genetic MTC, multiple endocrine neoplasia type II. Our studies of chromosome 10 in DNA from MTC tumors failed to detect frequent loss of polymorphic DNA markers, suggesting that the genetic mechanisms involved in MTC development may be different from those for other inherited cancers such as retinoblastoma. During tumor progression of MTC, abnormalities develop in expression of the mature phenotype of the endocrine cell from which the tumor arises. In cell culture, chemical modulation or gene insertion can lead to partial correction of these defects in differentiation capacity by activating cellular signaling processes. These studies offer opportunities to dissect the molecular events that regulate endocrine cell differentiation, to determine the precise abnormalities that may underlie the initiation and tumor progression events in MTC and related cancers, and, thereby, to identify new targets for therapeutic intervention.

The short arm of chromosome 11 is a "hot spot" for hypermethylation in human neoplasia.

Inactivation of normally expressed genes may play a role in the formation and/or progression of human cancers. Methylation of cytosine in DNA could potentially participate in such alterations of gene expression. Abnormalities in DNA methylation are a consistent feature of human neoplasms, and we now show that these include not only previously recognized widespread genomic hypomethylation, but also regional increases in gene methylation. A hot spot for abnormal methylation of C + G-rich areas has been detected on the short arm of chromosome 11 in an area known to harbor tumor suppressor genes. This change occurs consistently in common forms of human cancer and appears early during the transformation of cells with viruses including members of the human T-cell leukemia (HTLV) family. Furthermore, in one chromosome 11 gene examined, calcitonin, the increased methylation in somatic tumor cells coincides with the presence of an "inactive" chromatin pattern in the transcriptional regulatory area. The increased regional DNA methylation demonstrated may then participate in or mark chromosomal changes associated with gene inactivation events that are central to the genesis and/or progression of human cancers.

Transcriptional and posttranscriptional modulation of calcitonin gene expression by sodium n-butyrate in cultured human medullary thyroid carcinoma.

The TT cell line of human medullary thyroid carcinoma produces large quantities of calcitonin (CT) and calcitonin gene-related peptide (CGRP) mRNAs by alternative splicing of a primary CT gene transcript. We have previously shown that the relative levels of these mRNAs depend on the growth stages of the TT cells in culture and that these mRNAs can be increased acutely at the transcriptional level by phorbol esters (12-O-tetradecanoylphorbol-13-acetate) and the cyclic nucleotide, cyclic AMP. We show here that the naturally occurring fatty acid butyrate, unlike 12-O-tetradecanoylphorbol-13-acetate or cyclic AMP, has a delayed stimulatory effect on CT gene transcription, and also can modulate the posttranscriptional processing of RNA from this gene. Treatment of the TT cells with butyrate leads to a 5-fold increase in CT gene transcription after a lag period of 48 h and to a sustained decrease in the calcitonin gene-related peptide to CT mRNA ratio throughout the growth curve of these cells. In addition to its effects on CT gene expression, butyrate also decreases cellular proliferation and c-myc expression in the TT cells. These changes suggest that butyrate induces cultured human medullary thyroid carcinoma cells to acquire in vitro properties more consistent with the differentiated phenotype of the mature thyroid C-cell which is characterized by a low calcitonin gene-related peptide to CT ratio.

Hypermethylation of the 5' region of the calcitonin gene is a property of human lymphoid and acute myeloid malignancies.

An abnormal increase in numbers of CCGG sites methylated in the 5' region of the human calcitonin (CT) gene occurred in tumor cell DNA samples from 90% (17 of 19) of patients with non-Hodgkin's T and B cell lymphoid neoplasms and in 95% (21 of 22) of tumor cell DNA samples from patients with acute nonlymphocytic leukemia (ANLL). The changes were not seen in patients with chronic myelogenous leukemia (0 of 9). The abnormal methylation patterns appear to be a property only of transformed or malignant cells since they were not found in DNA from nonneoplastic adult tissues including sperm, early myeloid progenitor cells, benign lymphoid hyperplasia, peripheral lymphocytes stimulated to divide, or early myeloid progenitor cells (obtained by immunoaffinity using anti-My-10 antibody), but they did appear after Epstein-Barr virus transformation of lymphocytes. Moreover, during the course of therapy in patients with ANLL, the hypermethylation pattern reflects the presence of the leukemic clone even in normal-appearing granulocytes derived from this clone. The increased methylation of the CT gene may then provide an important molecular marker for biologic events in human cell transformation or tumor progression and may prove clinically useful in monitoring patients with lymphoid and acute myelogenous neoplasms.

Introduction of v-Ha-ras oncogene induces differentiation of cultured human medullary thyroid carcinoma cells.

Medullary thyroid carcinoma (MTC) is an endocrine tumor of the thyroid C cells that expresses high levels of the neuroendocrine peptide hormone calcitonin. During tumor progression in the host, there is an apparent loss of differentiation in MTC cells that involves a consistent decrease in calcitonin content of the tumor cells associated with decreased expression of the calcitonin gene and/or changes in a mRNA alternative-processing pattern away from that characteristic of the parent thyroid C cell. We now report that introduction of the viral Harvey ras (v-Ha-ras) oncogene into cultured human MTC cells can reverse such changes in gene expression and can induce endocrine differentiation of the tumor cells. The expression of v-Ha-ras is associated with decreased cellular proliferation and DNA synthesis. There is a marked increase in the number of cytoplasmic secretory granules that are a classic feature of differentiated thyroid C cells. v-Ha-ras expression induces increased expression of the calcitonin gene and the processing of the primary gene transcript is shifted to favor calcitonin mRNA rather than calcitonin-gene-related peptide (CGRP) mRNA production. These studies with cultured human MTC cells provide a model system to study the role of Ha-ras and related genes in neuroendocrine differentiation. The findings suggest an important approach for identifying genes in solid tumors whose altered expression may play a role in the impaired maturational capacity characteristic of cancer cells during tumor progression.

DNA methylation patterns of the calcitonin gene in human lung cancers and lymphomas.

Generalized hypomethylation of the genome and of specific genes has been described in human tumors. We now report that in human lung cancers, especially in the most aggressive form, small cell lung carcinoma, and in lymphomas, the 5'-region of the calcitonin (CT) gene exhibits methylation of increased numbers of CCGG sites in comparison with normal adult tissues. These unusual methylation patterns are found much less frequently in other tumor types examined. In the spectrum of the four major types of lung cancer (small cell, adeno-, squamous, and large cell carcinomas), the frequency of occurrence of hypermethylation in the 5'-region of the CT gene parallels that for presence of the neuroendocrine related biochemistry which characterizes small cell lung carcinoma. In medullary thyroid carcinoma, a tumor which expresses high levels of CT gene mRNA, the 5'-region of the CT gene is hypomethylated. Our findings provide a potential new molecular marker for two important human cancers (lung cancer and lymphomas) and suggest that there is a close relationship between abnormal CT gene methylation and developmental events for these tumors.