COMMEMORATIVE LECTURE
Isolation of disease-associated genes through genome analysis
and their clinical application
Yusuke Nakamura
Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science
The University of Tokyo, Tokyo, Japan(Received for publication on May 1, 2001)

Key words: Human Genome, Positional Cloning, p53, APC, VNTR (variable number of tandem repeat)

Thank you very much. Since I'm not the presidentof a university like Dr. Levine, I cannot talk for 30
minutes without any slides. Today, I am going to talkabout what I have done so far, what is the goal of myresearch, and what I would like to achieve. In the latter
half of my speech I would like to present you somespeci®c research topics I am working on.
As some of you may know, I had worked as an abdominal surgeon for four years after my graduationfrom Osaka University, Medical School. I was wondering at that time, what makes cancer cells moreprogressive? What makes the differences in individual
responses with regard to anti-cancer agents? Why are
they effective to some patients, but not to others, andwhy they cause very severe side effects and kill somepatients?
What I remember more when I worked as an abdominal surgeon are the patients who died of cancer.
Figure 1 shows a little poem written in the diary of oneof such patients, saying her appreciation to my assistance for her walking in the hallway. This patient who
had cancer had been my patient for two years. The
chemotherapy of a combination of anti-cancer drugs
worked effectively in the beginning, but later became
resistant to the therapy, and ®nally the patient died. I
still now regret whether I was able to provide suf®cientpsychological support to this patient as well as other
patients who died of cancer. What I learned from thesepatients is that cancer is a very serious disease, not only
for the patient but for the family members as well. The
questions and frustrations that I had during the four
years of clinical practice are my motivation to work
hard to control the deleterious disease, cancer.

As Dr. Levine said, dozens of genes involved incancer were found, and it is proven that cancer is a disease caused by genetic alterations. I had the good opportunity to study genetics at Dr. Ray White's laboratory, Howard Hughes Medical Institute, University of
Utah, where I worked on construction of human chromosomal genetic map as well as a gene responsible for
FAP, Familial Adenomatous Polyposis. Before going to
the USA, I read Dr. White's paper describing the method
to isolate a gene responsible for genetic diseases andfelt that it was very simple and easy. However, after
moving to Utah, I realized that the number of polymorphic genetic markers was not suf®cient enough toperform such studies. Hence, I was involved in the
project to isolate polymorphic markers to constructgenetic linkage maps. Figure 2 shows the result ofSouthern analysis using one of polymorphic variable
number of tandem repeats (VNTR) markers.1 This
marker is also useful in forensic science, and paternity
or maternity test as you can see how the chromosomes
are transferred from parents to children and to grandchildren. Using hundreds of these markers, we wereable to construct genetic maps for all human chromosomes that contributed enormously to the progressof studies on genetic diseases.2±5 Figure 3 shows thegenetic linkage map of human chromosome 10 consisting of 30 markers that I reported. This map was the best
genetic map at that time and all but three markers usedin this map were isolated by my group.
In June this year, Cerela and the international collaborative groups announced that over 90% of DNA
sequences of the human genome were determined. Ten
years ago, scientists had the chromosomal maps andPresented at the commemorative lecture of the award ceremony of the 2000 Keio Medical Science Prize, November 29, 2000.
Reprint requests to: Dr. Yusuke Nakamura, Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The
University of Tokyo, Shiroganedai 4-6-1, Minato-ku, Tokyo 108-8639, Japan134

Keio J Med 2001; 50 (3): 134±140

Fig. 1

A little poem written in the diary of one of my patients.

thought that it was very far and would take dozens ofyears to determine the entire DNA sequences of ourgenome. However, the technologies have so rapidly
progressed and we now have most of the genomic sequences although it takes several years to obtain 100%of the sequences with 100% accuracy. During my research in Utah, we found in collaboration with Dr.
Vogelstein that p53 was mutated in colorectal cancers
and had an important role in colorectal carcinogenesis.
The picture shown in this slide was taken in 1989, whenI ®rst met with Dr. Vogelstein in Japan although I often
talked with him on the phone when I was in the USA.
You can see one man behind Dr. Vogelstein and me inthis picture. This man is Dr. Yoshio Miki who ®rst discovered the BRCA1 gene, responsible for familial
breast and ovarian cancer.
As I mentioned, Dr, Vogelstein's and Dr. White'sgroups discovered p53 mutations in human cancers
11 years ago.6±10 After returning to Japan, I continued
to study familial adenomatous polyposis (FAP) andcloned the APC gene in collaboration with the Johns
Hopkins team in 1991.11±13 We also characterized frequent somatic mutations of the APC gene in sporadic
colorectal tumors and found the mutation cluster region(MCR) in the APC gene.14 Colorectal cancer is often
used as a model for multistep carcinogenesis as shownin this slide; Accumulation of mutations in APC, K-ras,

135

and p53 transform normal epithelial cells to adenoma
cells and then malignant colorectal cancer cells. However, I wondered how these discoveries contributed
to clinical studies or the clinical management. Except
some clinical test such as presymptomatic diagnosis forhereditary cancers, we have not been able to makegreat contributions.15±16Hence, we have been working hard to isolate additional genes associated with development and progression of cancer. I would like to show two examples. One
is the Axin gene. Dr. Yoichi Furukawa, an associate in
my group, discovered that the Axin gene was mutatedin hepatocellular carcinomas and had a tumor suppressive function in colorectal and liver cancers.17 We
have isolated a dozen of the candidate genes which
are involved in colorectal and hepatocellular carcinogenesis.
The other subject we have focused on is p53 that ismutated most frequently in human cancers. Although alarge numbers of papers related to p53 have beenreported, it is still not fully understood how it prevents
cancer, and how it kills cells. Dr. Hirofumi Arakawa
and his colleagues as well as Dr. Takashi Tokino, professor at Sapporo Medical College now, have been working on genes regulated by the wild-type p53.18±25 The
BAI1 and p53AIP1 genes, which we recently reported,
seem to have a potential to apply for treating cancer.
The BAI1 gene has a function to inhibit angiogenesisof cancer cells. We inoculated glioblastoma cells
subcutaneously into mice. Twelve days after the inoculation, we could see massive angiogenesis aroundthe cancer cells. However, we did not see any angiogenesis in the mice for which the cancer cells designed
to express the BAI1 gene were inoculated (Fig. 4).
The other gene, p53AIP1 (p53-dependent apoptosis
inducing gene) that we published in September, may
lead cancer cells to die.19 We inoculated human colorectal cancer cells at four regions in the back of a mouseand began treatment with p53AIP1 using the adenovirus vector after the cancer cells began to make a massof 3 mm diameter. The tumors without any treatment,
or treated with saline or with adenovirus designed toexpress LacZ became very large. However, the tumor
treated with p53AIP1 did not grow and completely disappeared three weeks later. We have so far tested thistherapy on nine mice, and in six of the nine mice thecancer cells have disappeared. Since the p53AIP1 gene
could kill some of the cancer cells which even p53
cannot kill, perhaps this treatment can be applied to avery wide range of cancer cells. We are now planning tohave studies leading this gene into clinical studies in the
future.
I have been working in the Human Genome Center,
The University of Tokyo and have been the Director of
the Center since 1995. From the human genome per-

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Nakamura Y: Medical importance of genome analysis

Fig. 2 Southern analysis using one of the polymorphic VNTR (variable number of tandem repeats) markers, YNH24, and a schematic presentation of its inheritance pattern in one family.

spective, we have a responsibility to train young
researchers who want to study in the human genome®eld. I accepted many doctors who were interested inthis ®eld, and some of them successfully isolated genes
responsible for genetic diseases or found the important
evidence that may be useful for clinical management ofpatients.26±30 Dr. Shiro Ikegawa, an orthopedic surgeon, was interested in the ectopic calci®cation andstudied on the mouse model called ttw (tip to walk).
Using the model showing a recessive trait, he successfully discovered that this abnormality was caused by theabnormal metabolism of phosphate. He interestingly
found that when these ttw mice were fed with foodcontaining a high concentration of phosphate, they
showed an abnormal shape of ear due to abnormal calci®cation of ear cartilage while no abnormality wasobserved when they were fed with low phosphate/low
calcium food or high calcium/low phosphate food. In
Japan, there are a large number of patients who haveossi®cation in spinal ligament (OPLL), our results could
lead to ®nd the molecular pathogenesis underlying thisdisease and may contribute to develop the novel treatment or chemoprevention for it.

I'm very sorry to show you a shocking picture (Fig. 5)of an eye affected by gelatinous drop-like corneal dystrophy. This disease is an autosomal recessive hereditary disorder and its prevalence is 1 in 300,000 people. The only one method to treat this disease istransplantation of cornea, but several years later thepatients' eyes usually show the same symptom. In orderto maintain their vision, they have to continuously
search for donations of cornea. Since this condition isvery miserable, it is essential to ®nd out the cause ofthis deleterious condition and develop a novel treatment. By cooperation with the patients and their family
members, we successfully identi®ed a gene responsible
for this disease and characterized their mutations. I
believe that the discovery of the causative gene shouldbe the ®rst step toward the development of a novel
treatment.
Although the results shown in the previous slides
contributed the better understanding of the etiology ofvarious diseases including cancer, it is still very dif®cult
to apply the results into the clinical ®eld. However, theresults we obtained may have a great possibility to prevent sudden cardiac death of patients with long QT

Keio J Med 2001; 50 (3): 134±140137Fig. 5 Eye affected by gelatinous drop-like corneal dystrophy andcharacterization of the mutations found in Japanese patients.

Fig. 3 A genetic linkage map of human chromosome 10 consisting of
30 polymorphic markers.

Fig. 4 Inhibition of angiogenesis induced in glioblastoma cells bymeans of ex vivo gene transfer of BAI1.

syndrome. The patients are characterized prolongationof the QT interval, leading to ventricular tachycardia
and syncopal attacks, sometimes sudden death. The bblocker is the conventional medicine used against thiscondition, but while roughly 70% of the patients respond well to this medicine, the remaining 30% of thepatients are non-responders. The genes responsible forthis condition were isolated and shown to be heterogeneous. Dr. Toshihiro Tanaka and his colleagues in
my laboratory screened mutations of these responsible
genes in more than 100 Japanese families carrying thelong QT syndrome and found that the causative genes
are associated with the ef®cacy of the b-blocker. His
results suggested that if they have a mutation in the
LQT1 gene, the patients should be treated with the bblocker alone, that if they have a mutation in the LQT2
gene, the patients should have a drug to prevent
arrhythmia or pacemaker implantation in addition to
the b-blocker. Therefore, identi®cation of the causative
gene may enable one to explain who are responders
and who are non-responders, and we may be able
to apply the personalized therapy that means a very
selective and the most appropriate therapy to a particular patient.
I always feel, because I was an abdominal surgeon,that I really want to provide a better treatment tocancer patients. Cancer is a very special subject for me.
In 1999, nearly 270,000 Japanese people died of cancer,
and 600,000 new people are found to have cancer
every year. At the advanced stage, the majority of thecancer patients are treated with chemotherapy without
knowing its effectiveness prior to the treatment. I'mvery sorry to say that the chemotherapy is equivalent to
gun shooting in the dark. In average, chemotherapy isjust effective in only 20% or 30% of the patients andthe remaining 70±80% of the patients suffer from theside effects without any improvements at all. It is apparent that it is not the right way to go. It is very important and essential to establish the method to predict

138

Nakamura Y: Medical importance of genome analysis

Fig. 8 A summary of the ef®cacy of nine anti-cancer drugs forten lung-cancer cell lines (the Central Institute for Experimental
Animals). The number ``100'' indicates that the sizes of the tumors
treated or untreated with anti-cancer drug were same and that the
drug was not effective to the cancer cells. On the other hand, ``zero''means that the cancer cells disappeared in this particular mouse, suggesting that this particular drug was very effective.
Fig. 6 A representative result of the expression pro®le of about
20,000 genes for AML.

Fig. 9 A representative result showing the signi®cant positive correlation between the expression level of C8098 and the ef®cacy to anticancer drug (CPM).
Fig. 7 Expression pro®les of 384 genes, a part of our cDNA collections for cDNA microarray analysis, in two colon-cancer lines in theupper and two glioblastoma cells lines in the lower.

the sensitivity to chemotherapy prior to starting thetreatment.
For this big challenge, we need to characterize indetail the nature of cancer cells and I thought that thesystematic analysis of expression pro®le may provide ananswer to us. Figure 6 shows the representative resultof the expression pro®le of about 23,000 genes for
AML. Green, red and yellow colors indicate the degreeof expression levels of each gene that corresponded toeach spot. Using the expression pro®les of cancer cells,
I believe that we will be able to characterize the different nature of cancer cells. For example, Figure 7 showsthe expression pro®le of 384 genes, a part of our cDNA
collections for cDNA microarray analysis, in two colon
tumor lines in the upper and two glioblastoma cell lines
in the lower. You can compare the expression pro®lesof two colon cancers and notice that the patterns of thetwo colon cancer cells are very similar, but very different from those of two glioblastoma cells. However, if
you look very carefully and compare the expression
patterns of the two colon cancer cells, you should notice
differences in the colors between the two. These subtle
differences may re¯ect the differences in the characterand properties of different cancers and some of thesedifferences may be associated with the responsivenessagainst anti-cancer drugs. Using the computer analysis,
we may be able to distinguish cancers that respondpoorly to anti-cancer drugs from those that respondwell.
We have been taken various approaches to ®ndgenes associated with drug ef®cacy using cancer celllines, xenografts, and also clinical cases. We have been
examining the correlations between the expression
pro®les and the sensitivities to each of nine different
anti-cancer drugs using xenograft materials in collabo-

Keio J Med 2001; 50 (3): 134±140

ration with the Central Institute for Experimental Animals. Cancer cells were inoculated into nude mice andthen treated with each of the nine anti-cancer drugs,
MMC, CPM, ACNU, DDP, ADR, VCR, VLB, 5-FU,
and MTX. Two weeks later, the sizes of the tumors
were measured and the ef®cacy of each drug was estimated. Figure 8 presents a summary of the experiments. The number ``100'' indicates that the sizes of thetumors treated or untreated with anti-cancer drug were
the same and that the drug was not effective to thecancer cells. On the other hand, ``zero'' means that thecancer cells disappeared in this particular mouse, suggesting that this particular drug was very effective. We
then analyzed the expression levels of each of the
23,000 genes using the cDNA microarray. Figure 9shows the representative result showing a signi®cant
positive correlation. As you can see, the expression
levels indicated by the different colors clearly correlated with the sensitivity to this particular anti-cancer
drug. Currently, we completely ®nished the expression
analysis of the 90 cancer cell lines and picked up hundreds of genes showing some positive association withthe effectiveness of each of the nine anti-cancer drugs.
I would also like to show the results using clinicalmaterials. Expression pro®les of tissues obtained from
20 patients, who had been operated on for esophageal
cancer, all at stage III or stage IV advanced cancer,
were analyzed. Most of the patients had a non-curative
operation and were con®rmed to have residual cancer
cells microscopically or macroscopically. After surgicaltreatment, all of the patients had chemotherapy of a
combination of cisplatin and 5-FU. We divided thesepatients into three different classes on the basis of theirsurvival period after the surgical operation; eightpatients who survived more than 30 months were classi®ed as group 1, six who died within 12±30 months after the operation as group 2, and those who died within
12 months as group 3. Since the main tumor in theesophagus had been resected, we cannot say that thesurvival length re¯ected the ef®cacy of chemotherapy.
However, we can speculate that this longer survival
may likely be related to the ef®cacy of chemotherapy
and for the patients who died within one year, it is very
likely that chemotherapy was not effective at all.
The nature of the cancer cells is very likely to be
determined by the combination of the genes expressedin the cell. We attempted to compare the expression
pro®les between the tumors belonging to group 1 andgroup 3. The expression patterns seem to be quite similar, but there were substantial differences in the expression levels of some genes. We then analyzed all thedata and picked up 52 genes whose expression levels
may be correlated with the prognosis of these patients.
cDNA spots corresponding to some genes were commonly red in tumors belonging to group 1, but yellow or139

green in those to group 3. Some showed the inverted
patterns, indicating the strong correlation between theexpression levels of these genes and the prognosisof the patients. Furthermore, to apply these results toclinical diagnosis, we convert these color patterns tonumerical scores by means of algorithms we established. The three groups were clearly separated by the
drug response score (DRS), indicating that this scoring
system may have a great potential to establish themethod to predict the sensitivity to anti-cancer drugs.
To further clarify the scoring system, we obtained andexamined DRSs of tumors of seven additional cases.
The results of these seven cases were consistent withthose of the original test cases without any exception.
The goal of my approach is summarized in the last slide.
I would like to accumulate the information of expression pro®les and clinicopathological data from a large
number of clinical cases and establish the database thatcan predict the most appropriate treatment for a particular patient. We should establish the personalized
medicine using genetic or genomic information, ``anappropriate dose of the right drug to the right patient''.
I thank all of my co-workers for helping my researchand it is sure that I could not receive the Keio Medical
Prize, the very prestigious prize, without their joining to
my laboratory.
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