1. Healthcare

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4388
CORRESPONDENCE
platelet count. However, taking into account the somewhat weaker
thrombopoietic activity of Epo in humans11 as compared with mice, it
remains to be seen whether Epo could be effective in correcting
IL-12–induced thrombocytopenia in tumor patients.
Because hematopoietic growth factors may occasionally stimulate
the growth of neoplasms, including solid tumors,12,13 we decided to
examine the influence of Epo application on the growth of MmB16
melanoma. Epo did not influence tumor growth when given alone and
did not decrease the antitumor activity of IL-12 in this particular tumor
model (Fig 1A and B).
As the antitumor activity of IL-12 may by potentiated by administration of some of the chemotherapeutics,14 it might be anticipated that
IL-12 together with Epo could be successfully included into some of the
combined chemoimmunotherapy schedules.
ACKNOWLEDGMENT
Supported by Grant No. 6 P207 058 07 from the State Committee for
Scientific Research (K.B.N), Poland. Tomasz Stokłosa is a recipient of
the Foundation for Polish Science Award.
Jakub Goła¸b
Radosław Zagoz˙dz˙on
Tomasz Stokłosa
Witold Lasek
Marek Jako´bisiak
Department of Immunology
Institute of Biostructure
Medical University of Warsaw, Poland
Zygmunt Pojda
Department of Radiation Hematology
WIHiE, Warsaw, Poland
Eugeniusz Machaj
Department of Experimental Hematology
Maria Skłodowska-Curie Memorial Cancer Center
Institute of Oncology, Warsaw, Poland
REFERENCES
1. Nastala CL, Edington HD, McKinney TG, Tahara H, Nalesnik
MA, Brunda MJ, Gately MK, Wolf SF, Schreiber RD, Storkus WJ,
Lotze MT: Recombinant IL-12 administration induces tumor regression
in association with IFN-g production. J Immunol 153:1697, 1994
2. Lasek W, Feleszko W, Goła¸b J, Stokłosa T, Marczak M, Da¸browska A, Malejczyk M, Jako´bisiak M: Antitumor effects of the
combination immunotherapy with IL-12 and TNF-a in mice. Cancer
Immunol Immunother 45:100, 1997
3. Jacobsen SEW, Veiby OP, Smeland EB: Cytotoxic lymphocyte
maturation factor (Interleukin 12) is a synergistic growth factor for
hematopoietic stem cells. J Exp Med 178:413, 1993
4. Eng VM, Car BD, Schnyder B, Lorenz M, Lugli S, Aguet M,
Anderson TD, Ryffel B, Quesniaux VFJ: The stimulatory effects of
interleukin (IL)-12 on hematopoiesis are antagonized by IL-12-induced
interferon g in vivo. J Exp Med 181:1893, 1995
5. Atkins MB, Robertson MJ, Gordon M, Lotze MT, DeCoste M,
DuBois JS, Ritz J, Sandler AB, Edington HD, Garzone PD, Mier JW,
Canning CM, Battiato L, Tahara H, Sherman ML: Phase I evaluation of
intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 3:409, 1997
6. Coughlin CM, Wysocka M, Trinchieri G, Lee WMF: The effect of
interleukin 12 desensitization on the antitumor effects of recombinant
interleukin 12. Cancer Res 57:2460, 1997
7. Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G,
Atkins MB, Sosman JA, Dutcher JP, Vogelzang NJ, Ryan JL: Effects of
single-dose interleukin-12 exposure on interleukin-12–associated toxicity and interferon-g production. Blood 90:2541, 1997
8. Goła¸b J, Stokłosa T, Zagoz˙dz˙on R, Kaca A, Giermasz A, Pojda Z,
Machaj E, Da¸browska A, Feleszko W, Lasek W, Iwan-Osiecka A,
Jako´bisiak M: G-CSF prevents the suppression of bone marrow
hematopoiesis induced by IL-12 and augments its antitumor activity in
melanoma model in mice. Ann Oncol 9:63, 1998
9. Spivak JL: Recombinant human erythropoietin and the anemia of
cancer. Blood 84:997, 1995
10. Harrison J, Kappas A, Levere RD, Lutton JD, Chertkov JL, Jiang
S, Abraham NG: Additive effect of erythropoietin and heme on murine
hematopoietic recovery after azidothymidine treatment. Blood 82:3574,
1993
11. Eschbach JW, Abdulhadi MH, Browne JK, Delano BG, Downing
MR, Egrie JC, Evans RW, Friedman EA, Graber SE, Haley NR, Korbet
S, Krantz SB, Lundin AP, Nissenson AR, Ogden DA, Paganini EP,
Rader B, Rutsky EA, Stivelman J, Stone WJ, Teschan P, van Stone JC,
van Wyck DB, Zuckerman K, Adamson JW: Recombinant human
erythropoietin in anemic patients with end-stage renal disease. Results
of a phase III multicenter clinical trial. Ann Intern Med 111:992, 1989
12. Segawa K, Ueno Y, Kataoka: In vivo tumor growth enhancement
by granulocyte colony-stimulating factor. Jpn J Cancer Res 82:440,
1991
13. Feleszko W, Giermasz A, Goła¸b J, Lasek W, Kuc K, Szperl M,
Jako´bisiak M: Granulocyte-macrophage colony-stimulating factor accelerates growth of Lewis lung carcinoma in mice. Cancer Lett 101:193,
1996
14. Zagoz˙dz˙on R, Stokłosa T, Goła¸b J, Giermasz A, Kaca A,
Kultchitska LA, Lasek W, Jako´bisiak ML: Combination tumor therapy
with interleukin-12, cisplatin, and tumor necrosis factor-a of experimental melanoma in mice. Anticancer Res 17:4493, 1997
15. Lasek W, Giermasz A, Kuc K, Wan´kowicz A, Goła¸b J, Zagoz˙dz˙on
R, Stokłosa T, Jako´bisiak M: Potentiation of the antitumor effect of
actinomycin D by tumor necrosis factor a in mice: Correlation between
results of in vitro and in vivo studies. Int J Cancer 66:374, 1996
Alterations of the MMAC1/PTEN Gene in Lymphoid Malignancies
To the Editor:
The MMAC1/PTEN gene is localized on chromosome 10q23.3 and
encodes a putative tumor suppressor with structural homologies to
known phosphatases and cytoskeletal proteins.1,2 Functional studies
have shown that MMAC1/PTEN is a dual-specificity protein phosphatase that dephosphorylates tyrosine, serine, and threonine.3,4 Germline
mutations of MMAC1/PTEN have been implicated as the predisposing
factor in Cowden disease,5 and loss of function mutations have been
found in a variety of sporadic solid cancers.1,2,6 Previous observations of
10q abnormalities in lymphoproliferative diseases7,8 suggest that
MMAC1/PTEN may possibly be involved in lymphomagenesis.
We systematically studied 14 malignant and 4 benign lymphoid cell
lines for deletions and mutations in all exons of the MMAC1/PTEN gene
by combining polymerase chain reaction (PCR), denaturing gradient gel
electrophoresis (DGGE) and direct sequence analysis.9 Overall, alterations of MMAC1/PTEN were shown in 2 of the malignant cell lines and
in none of the benign cell lines. The T-cell acute lymphoblastic
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
CORRESPONDENCE
leukemia cell line CCRF-CEM harbored homozygous deletion of a
genomic region including exons 2 through 5. In the myeloma cell line
HS-Sultan, we identified a C = T transition at the first base of codon
17, resulting in the substitution of glutamine with a premature
termination signal. This cell line also showed loss of the wild-type
allele.
We next examined all exons of the MMAC1/PTEN gene by PCR/
DGGE analysis in 170 primary lymphoid malignancies (Fig 1). This
approach led to the detection of point mutations in 2 (5%) of 39 diffuse
large B-cell lymphomas. One of the samples harbored a G = C
transversion at the third base of codon 342, which is predicted to cause
the substitution of lysine with asparagine. This mutation affects the last
base of exon 8, which is considered to be part of the donor-splice site
consensus sequence required for correct splicing of pre-mRNA.10
Several examples of mutations at position 21 of donor-splice sites that
cause skipping of the preceding exon have been reported.11 Whether the
MMAC1/PTEN Lys342Asn mutation has functional consequences with
respect to splicing remains to be investigated. Analysis of normal tissue
from the patient showed only the normal sequence, documenting that
the mutation occurred somatically. The second sample harbored a
G = A transition at the second base of codon 10, causing substitution of
serine with asparagine. This residue is conserved in auxilin and the
hypothetical yeast PTPase, YNL128W.1 Semiquantitative DGGE analysis9 showed unequal distribution of mutant and wild-type sequences,
suggesting that the mutation was present in only a subpopulation of
cells. None of the mutations we identified have been reported to occur in
normal tissues.12
Although MMAC1/PTEN alterations were found in only a minority of
lymphoma cell lines and tumor samples, our results, together with the
recent demonstration of mutations in leukemia cell lines,6 indicate that
MMAC1/PTEN may be a target in the pathogenesis or progression of
some forms of hematological malignancy.
Fig 1. Detection and identification of MMAC1/PTEN mutations in
primary lymphomas. (A) PCR/DGGE analysis of MMAC1/PTEN exon 1
in 10 tumor samples. (B) Direct sequence analysis of the sample
displaying an aberrant band pattern in (A). The partial sequence
ladder shows a G = A transition, resulting in the substitution of Ser10
with Asn10.
4389
ACKNOWLEDGMENT
We are grateful to Dr H.E. Johnsen who generously provided cell
lines, and to V. Ahrenkiel and L. Jensen for expert technical assistance.
Supported by grants from the Danish Cancer Society, Kong Christian
X’s Foundation, the Hasselbalch Foundation, and Søeborg Ohlsens
Foundation.
Kirsten Grønbæk
Department of Tumor Cell Biology
Institute of Cancer Biology
Danish Cancer Society
Copenhagen, Denmark
Department of Pathology
Herlev Hospital
Copenhagen, Denmark
Jesper Zeuthen
Per Guldberg
Department of Tumor Cell Biology
Institute of Cancer Biology
Danish Cancer Society
Copenhagen, Denmark
Elisabeth Ralfkiær
Department of Pathology
Herlev Hospital
Copenhagen, Denmark
Klaus Hou-Jensen
Department of Pathology
Rigshospitalet
Copenhagen, Denmark
REFERENCES
1. Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH,
Langford LA, Baumgard ML, Hattier T, Davis T, Frye C, Hu R,
Swedlund B, Teng DH, Tavtigian SV: Identification of a candidate
tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is
mutated in multiple advanced cancers. Nat Genet 15:356, 1997
2. Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J,
Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC,
Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R: PTEN, a
putative protein tyrosine phosphatase gene mutated in human brain,
breast, and prostate cancer. Science 275:1943, 1997
3. Li D-M, Sun H: TEP1, encoded by a candidate tumor suppressor
locus, is a novel protein tyrosine phosphatase regulated by transforming
growth factor b. Cancer Res 57:2124, 1997
4. Myers MP, Stolarov JP, Eng C, Li J, Wang SI, Wigler MH, Parsons
R, Tonks NK: P-TEN, the tumor suppressor from human chromosome
10q23, is a dual-specificity phosphatase. Proc Natl Acad Sci USA
94:9052, 1997
5. Liaw D, Marsh DJ, Li J, Dahia PL, Wang SI, Zheng Z, Bose S,
Call KM, Tsou HC, Peacocke M, Eng C, Parsons R: Germline mutations
of the PTEN gene in Cowden disease, an inherited breast and thyroid
cancer syndrome. Nat Genet 16:64, 1997
6. Teng DH, Hu R, Lin H, Davis T, Iliev D, Frye C, Swedlund B,
Hansen KL, Vinson VL, Gumpper KL, Ellis L, El-Naggar A, Frazier M,
Jasser S, Langford LA, Lee J, Mills GB, Pershouse MA, Pollack RE,
Tornos C, Troncoso P, Yung WK, Fujii G, Berson A, Steck PA:
MMAC1/PTEN mutations in primary tumor specimens and tumor cell
lines. Cancer Res 57:5221, 1997
7. Speaks SL, Sanger WG, Masih AS, Harrington DS, Hess M,
Armitage JO: Recurrent abnormalities of chromosome bands 10q23-25
in non- Hodgkin’s lymphoma. Genes Chromosomes Cancer 5:239,
1992
8. Takeuchi S, Bartram CR, Wada M, Reiter A, Hatta Y, Seriu T, Lee
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4390
CORRESPONDENCE
E, Miller CW, Miyoshi I, Koeffler HP: Allelotype analysis of childhood
acute lymphoblastic leukemia. Cancer Res 55:5377, 1995
9. Guldberg P, thor Straten P, Birck A, Ahrenkiel V, Kirkin AF,
Zeuthen J: Disruption of the MMAC1/PTEN gene by deletion or
mutation is a frequent event in malignant melanoma. Cancer Res
57:3660, 1997
10. Padgett RA, Grabowski PJ, Konarska MM, Seiler S, Sharp PA:
Splicing of messenger RNA precursors. Annu Rev Biochem 55:1119,
1986
11. Krawczak M, Reiss J, Cooper DN: The mutational spectrum of
single base-pair substitutions in mRNA splice junctions of human
genes: Causes and consequences. Hum Genet 90:41, 1992
12. Tsou HC, Teng DH, Ping XL, Brancolini V, Davis T, Hu R, Xie
XX, Gruener AC, Schrager CA, Christiano AM, Eng C, Steck P, Ott J,
Tavtigian SV, Peacocke M: The role of MMAC1 mutations in earlyonset breast cancer: Causative in association with Cowden syndrome
and excluded in BRCA1-negative cases. Am J Hum Genet 61:1036,
1997
A Response to AC133 Hematopoietic Stem Cell Antigen: Human Homologue of Mouse Kidney
Prominin or Distinct Member of a Novel Protein Family?
Three recently published works represent the first descriptions of a
potential new family of 5-trans-membrane cell surface glycoproteins.1,2,3 The human AC133 molecule2,3 may indeed be the homologue
of mouse prominin,1,4 and Drs Weigmann and colleagues have very
carefully discussed the evidence for and against this relationship. We
agree with the arguments put forward by these authors, and that further
work is clearly needed to examine the possible localization of AC133
antigen to plasma membrane protrusions, and to assess the expression
of prominin on murine hematopoietic stem cells.
If prominin proves to be a marker of primitive murine stem cells, as
AC133 is in humans, its value in studies of murine hematopoiesis would
be considerable. To assist these studies, we have also isolated and
characterized a prominin-like cDNA from mouse brain. This murine
sequence differs slightly from the prominin sequence, as we have
identified an additional block of nine amino acids within the N-terminal
domain. These nine amino acids are strongly homologous to the
corresponding human sequence (Fig 1). The cDNA isolated from
mouse brain therefore predicts a protein of 867 amino acids, with a
Fig 1. Alignment of human AC133, mouse AC133, and mouse
prominin sequences, showing the position of a presumed nine–
amino acid deletion in the mouse prominin sequence.
molecular weight of 97.3 kD.5 The presence of murine AC133 transcript
in a variety of mouse tissues was assessed by Northern analysis (Fig 2).
A 4.4-k mRNA transcript was detectable in mouse embryo at 7 days,
peaking at 11 days, and tapering off at days 15 and 17 while still being
clearly detectable. In adult mouse tissue, the transcript is weakly
detectable in brain, lung, skeletal muscle, and more intensely in kidney
and testis. Molecular analysis of the three proteins from human, mouse
and Caenorhabtidis elegans6 indicate that all three contain leucine
zipper consensus sequences (on the first extracellular loop in the human
and mouse, and on the second in C elegans). There are conserved
cysteine residues in all three molecules appearing at the beginning of
each small cytoplasmic loop. Although the mouse and human proteins
exhibit strong conservation in all five predicted transmembrane sequences, the greatest degree of conservation with the C elegans
sequence is within TM3, where 7 out of 22 amino acids are conserved in
all three species. There are seven tyrosine residues within the C-terminal
cytoplasmic tail in C elegans; however, none of them occur within a
tyrosine phosphorylation consensus sequence. The cytoplasmic tails of
the mouse and human proteins contain five tyrosine residues, the first of
which is a tyrosine phosphorylation consensus sequence that is completely identical in both proteins. Another sequence7 in the Expressed
Sequence Tag database, derived from murine embryo, has approximately 25% homology to the human and mouse AC133 antigen/
prominin sequences, indicating that these two molecules may indeed be
the first described of a new family of cell surface receptors.
The expression of such a gene or gene family on hematopoeitic
human stem cells, mouse embryo, and adult tissues, and in a phylogenetically less evolved organism such as C elegans may indicate an
important role for this five-transmembrane protein structure in development and/or cell signaling. In particular, the localization of the prominin
molecule to cellular protrusions places this molecule in an optimal
Fig 2. Northern analysis of murine AC133 antigen
mRNA expression. Multiple tissue poly A1 Northern
blots were purchased from Clontech (San Diego, CA)
and probed with 32P murine AC133 antigen cDNA. (A)
Expression of message is detectable in mouse embryo peaking at day 11, and then tapering off to still
detectable levels at days 15 and 17. (B) In adult
tissues, message is detectable in brain, lung, skeletal
muscle, kidney, and testis.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1998 91: 4388-4390
Alterations of the MMAC1/PTEN Gene in Lymphoid Malignancies
Kirsten Grønbæk, Jesper Zeuthen, Per Guldberg, Elisabeth Ralfkiær and Klaus Hou-Jensen
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