Faculty Bibliography

It has been hypothesized that the early acquisition of genomic instability is essential to account for the high number of mutations commonly identified in human cancers. Previous reports have demonstrated that MYC overexpression results in gene amplification. Given the importance of MYC in Burkitt's lymphoma, we investigated the effects of MYC on spontaneous inactivating somatic mutations, using an assay that we have developed, based on the PIG-A gene. PIG-A is X-linked, thus a single mutation can disrupt its function. PIG-A encodes an enzyme required for the biosynthesis of glycosylphosphatidylinositol (GPI), which is required for the expression of a set of membrane proteins on the cell surface (e.g., CD48, CD52, CD55, and CD59). Expansion of PIG-A mutant stem cells occurs in PNH, and it is known from this disorder that a very broad spectrum of mutations can inactive PIG-A. Using PIG-A, we have previously determined the mutation rate (mu ) in EBV transformed B-lymphoblastoid cell lines (BLCLs) from normal human donors, and we have demonstrated genomic instability in cell lines derived from hematologic malignancies. Here we have measured mu in P493, a cell line derived from normal human B cells, which overexpress MYC (normalized levels 17 x higher than BLCLs). P493 can also be induced to express lower levels of MYC, resulting in a lower growth rate, using tetracycline and estradiol. To measure mu, pre-existing mutants are first eliminated from the culture by staining the cells with an antibody specific for CD59 and sorting to collect the upper 50th percentile of the population. This purified GPI(+) population is then returned to culture for 3 weeks, with careful cell counts taken to estimate the number of cell divisions (d) occurring in vitro. After expansion, the frequency (f) of mutants arising in vitro is measured by first incubating with a mixture of mouse antibodies specific for CD48, CD52, CD55, and CD59, followed by a rabbit anti-mouse PE conjugate, followed by a FITC conjugated antibody specific for a transmembrane protein (CD45 or HLA-DR). Live cells are identified based on exclusion of propidium iodide and based on FITC fluorescence; GPI(-) cells are defined as having <4% of the mean PE fluorescence as the GPI(+) population. f is calculated as # GPI(-) cells/total gated live cells. The mutation rate is then calculated as mu = f/d. In initial experiments, 6 separate cultures of P493 were analyzed, demonstrating a median mu of 15 x 10^-7 mutations per cell division (range 11 - 33 x 10^-7). For comparison, the positive control, a hypermutable mantle cell lymphoma cell line (HBL2), demonstrated a mu of 674 x 10^-7, and five BLCLs derived from normal donors demonstrated a median mu value of 37 x 10^-7 (range 11 to 57 x 10^-7). In parallel experiments, P493 was also grown in the presence of tetracycline and estradiol to reduce MYC: in 5 separate experiments, P493 growing in these conditions demonstrated a median mu of 6 x 10^-7. However, in 2 subsequent experiments, we observed a higher mu under "lower MYC" conditions: 81 x 10^-7 (SEM +/-15 x 10^-7) vs 13 x 10^-7 (+/-2.2 x 10^-7) and 65 x 10^-7 (+/-6.2 x 10^-7) vs 21 x 10^-7(+/-3.3 x 10^-7). We then isolated 6 clones of P493: for 5 clones, under "high MYC" conditions, mu was similar to the previous experiments (26 x 10^-7, SEM +/-3.4 x 10^-7), and for 3 of these clones mu went up in the "lower MYC" conditions by 5 to 9 fold. For 2 of these clones, mu was similar in the "high MYC" and "lower MYC" conditions. One of the 6 clones, surprisingly, did not adopt a lower growth rate in the presence of tetracycline and estradiol and demonstrated a reversed pattern: mu was 70 x 10^-7 under "high MYC" conditions and 16 x 10^-7 under "lower MYC" conditions. We next analyzed 7 cell lines derived from Burkitt's neoplasms which also overexpressed MYC (mean normalized levels ~ 3 x higher compared with normal BLCLs). Here we found a bimodal pattern, as we have previously described for primary acute lymphoblastic leukemia samples: for 4 of the cell lines mu was < 25 x 10^-7, whereas 3 samples exhibited mu values comparable to the hypermutable positive control: 229 x 10^-7, 562 x 10^-7, and 1786 x 10^-7 respectively. We conclude that overexpression of MYC is not sufficient to produce hypermutability in human B cells, based on this assay for gene-inactivating mutations. Hypermutability-as detected here-is a common feature of cell lines derived from Burkitt's neoplasms but does not seem to be necessary for its development

Mutations are an inherent risk of cell duplication. On one hand, inheritable mutations are the driving force of biological evolution; on the other hand, their accumulation in somatic cells plays a key role in the development of cancer. The frequency of mutants (f) and the rate of mutation (mu) are biological features of any cell population: their measurement could provide important information about the risk of oncogenesis and the exposure to carcinogenic agents. However, the measurement of these parameters is not trivial. To measure f and mu, a potential sentinel gene is the PIG-A gene, which encodes one of the subunits of an enzyme essential in the biosynthesis of glycosylphosphatidylinositol (GPI). Since PIG-A is X-linked, mutational inactivation of the one single copy active in somatic cells entails absence from the cell surface of all the proteins that require GPI for attachment to the membrane: thus, mutant cells display a GPI-negative surface phenotype that can be easily detected by flow cytometry. The measurement of PIG-A mutants by counting cells with the GPI-negative phenotype has proved to be effective to measure mutant frequency in peripheral blood cells of humans and of others animals. Up to now, mu has been exceedingly difficult to measure in human cells; however, by using as a sentinel the PIG-A gene in lymphoblastoid cell lines we now have a test that makes it practical to measure mu in human cells

It has been proposed that hypermutability is necessary to account for the high frequency of mutations in cancer. However, historically, the mutation rate (mu) has been difficult to measure directly, and increased cell turnover or selection could provide an alternative explanation. We recently developed an assay for mu using PIG-A as a sentinel gene and estimated that its average value is 10.6 x 10(-7) mutations per cell division in B-lymphoblastoid cell lines (BLCLs) from normal donors. Here we have measured mu in human malignancies and found that it was elevated in cell lines derived from T cell acute lymphoblastic leukemia, mantle cell lymphoma, follicular lymphoma in transformed phase, and 2 plasma cell neoplasms. In contrast, mu was much lower in a marginal zone lymphoma cell line and 5 other plasma cell neoplasms. The highest mu value that we measured, 3286 x 10(-7), is 2 orders of magnitude above the range we have observed in non-malignant human cells. We conclude that the type of genomic instability detected in this assay is a common but not universal feature of hematologic malignancies

We describe a patient with paroxysmal nocturnal hemoglobinuria (PNH) and no previous history of thrombosis who presented with hepatic venous thromboses and subsequently developed splenic infarction and rupture requiring splenectomy while on anticoagulation therapy for the hepatic thromboses. The patient's anticoagulation was complicated by heparin-induced thrombocytopenia (HIT) highlighting the unique management challenge presented by PNH in combination with HIT.

Very few human genes can be used to identify spontaneous inactivating somatic mutations. We hypothesized that because the XK gene is X-linked, it would be easy to identify spontaneously arising red cells with a phenotype resembling the McLeod syndrome, which results from inherited XK mutations. Here, by flow cytometry, we detect such phenotypic variants at a median frequency of 9x10(?6) in neonatal cord blood samples and 39x10(?6) in healthy adults (p=0.004). It may be possible to further investigate the relationship between aging, mutations, and cancer using this approach