| Summary: | 博士 === 高雄醫學院 === 醫學研究所 === 86 === Leukemia and cancers are caused by accumulation of multiple
genetic alterations, including the gain function of proto-
oncogenes and the inactivation of tumor suppressor genes
controlling the cell cycle. Three novel genes were identified
recently at chromosome 3p14.2, 11p15.1-15.2, 10q23.3, named
FHIT, TSG101, PTEN/MMAC1, and they were designated as potential
human tumor suppressor genes. The FHIT gene encodes a 147 amino-
acid protein, dinucleoside 5'',5''''''-p1,p3-triphosphate (Ap3A)
hydrolase which is homologous to a group of proteins designated
HIT proteins. Ap3A has been proposed to have various
intracellular functions, including regulation of DNA replication
and signaling stress responses. The TSG101 is predicted to
encode a 380 amino-acid protein which contains a proline-rich
domain and DNA-binding motifs characteristic for a transcription
factor and a coiled-coil domain shown to interact with stathmin.
Stathmin is a cytoplasmic phosphoprotein which was elevated in a
variety of malignancies and proposed to coordinate and relay
diverse signals regulating cell proliferation and
differentiation. The PTEN/MMAC1 protein contains protein
tyrosine phosphatase domain and extensive homology to tensin, a
protein that interacts with actin filaments at focal adensions.
To determine the roles of the FHIT, TSG101, PTEN/MMAC1 genes in
leukemia, bone marrow or peripheral blood from myeloid leukemia
patinets and five hematopoietic cell lines (HL60, U937, Raji,
KG-1, K562) were analyzed by reverse transcription of the mRNA
followed by PCR amplification and sequencing of the products. To
detect the deletion of the gnens, 17 cases were evaluated using
microsatellite polymorphism analysis. In this study, 17/62(27%)
AML patients, 1/15(7%) CML patients in the chronic phase, and
3/18(16%) CML patients in the blastic phase expressed aberrant
FHIT transcripts which lack two or more exons of the FHIT
transcripts. 24/68 (35%) AML patietns, 2/15 (13%) CML patients
in the chronic phase, and 5/18 (28%) CML patients in the blastic
phase expressed aberrnat TSG101 transcripts. 15/60(25%) AML
patients, and 1/18(6%) CML patients in the blastic phase
expressed PTEN/MMAC1 aberrant transcripts. All the cell lines
expressed aberrant FHIT, TSG101, and PTEN/MMAC1 transcripts,
excepts K562 displayed only the normal PTEN/MMAC1 transcript. No
case exhibited a loss of the FHIT and PTEN/MMAC1 alleles. The
aberrant transcripts may result from alternative RNA splicing as
evidenced by normal and aberrant transcripts are found in all
specimens examined. The aberrant transcripts may cause the
reduction of the protein or affect the function of the protein,
and alter the control of the cell cycle. We construct a deleted
mutant form of internal standard to detect the expression of the
cell cycle related genes (cyclin D1, cyclin E, CDK2 and CDK4).
We add the specimens, internal standard into the same reaction
tube to perform the competitive polymerase chain reaction with
the specific primers. In our study, we can find the increasd
expression of cyclin D1, cyclin E, CDK2, and CDK4 in CML
patients from the chronic phase to the blastic crisis. We also
find the overexpression of the cell cycle related genes in our
AML patients. These results indicated that the cell cycle
related genes are overexpressed in myeloid leukemia, and may be
related to the progression of leukemia. We also studied
microsatellite instability in 9 CML patients with chronic and
blastic crisis phase, 18 AML patients, and 3 ALL patients with
diagnotic and relapse stage. Microsatellite are highly
polymorphic, short tandem repeat sequences throughout the
genome. Microsatellite instability are mutations of these repeat
sequences, which were resulted from mutation of DNA mismatch
repair genes (MSH2, MLH1, PMS1, PMS2). Three microsatellite
instability loci (D2S123, D5S299, D17S179) were found in one CML
patient with blastic phase, and four microsatllite instability
loci (D2S123, D5S299, D17S179, D18S34) were found in one AML and
one ALL patient with relapse stage. These results indicate that
the dysfunction of mismatch repair genes may be related to the
progression of myeloid leukemia. In colorectal cancer, the
dysfunction of the mistmath repair genes cause the genetic
instability, and accelerate the transformation from the normal
colonic epithelial tissue through adenoma to carcinoma which
appears to be accompanied by mutations in the APC, RAS, DCC, and
p53 genes. Through our study, the accumulation of multiple
genetic alterations may play an important role in myeloid
leukemia. We found the genetic instability in AML, ALL with
relapse stage, and CML with blastic phase. We also found
aberrant FHIT, TSG101, PTEN/MMAC1 transcripts, and it may affect
the control of the cell cycle and cell growth, which lead to the
formation of leukemia.
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